ABSTRACT: MicroRNAs in Amoebozoa: Deep sequencing of the small RNA population in the social amoeba Dictyostelium discoideum reveals developmentally regulated microRNAs
Project description:Small RNAs play crucial roles in regulation of gene expression in many eukaryotes. Here, we report the cloning and characterization of 18-26 nt RNAs in the social amoeba Dictyostelium discoideum. This survey uncovered developmentally regulated microRNA candidates whose biogenesis, at least in one case, is dependent on a Dicer homolog, DrnB. Furthermore, we identified a large number of 21 nt RNAs originating from the DIRS-1 retrotransposon, clusters of which have been suggested to constitute centromeres. Small RNAs from another retrotransposon, Skipper, were significantly up-regulated in strains depleted of the second Dicer-like protein, DrnA, and a putative RNA-dependent RNA polymerase, RrpC. In contrast, the expression of DIRS-1 small RNAs was not altered in any of the analyzed strains. This suggests the presence of multiple RNAi pathways in D. discoideum. In addition, we isolated several small RNAs with antisense complementarity to mRNAs. Three of these mRNAs are developmentally regulated. Interestingly, all three corresponding genes express longer antisense RNAs from which the small RNAs may originate. In at least one case, the longer antisense RNA is complementary to the spliced but not the unspliced pre-mRNA, indicating synthesis by an RNA-dependent RNA polymerase. Keywords: cDNA library; small RNA sequencing
Project description:ATP-binding cassette (ABC) transporters can translocate a broad spectrum of molecules across the cell membrane including physiological cargo and toxins. ABC transporters are known for the role they play in resistance towards anticancer agents in chemotherapy of cancer patients. There are 68 ABC transporters annotated in the genome of the social amoeba Dictyostelium discoideum. We have characterized more than half of these ABC transporters through a systematic study of mutations in their genes. We have analyzed morphological and transcriptional phenotypes for these mutants during growth and development and found that most of the mutants exhibited rather subtle phenotypes. A few of the genes may share physiological functions, as reflected in their transcriptional phenotypes. Since most of the abc-transporter mutants showed subtle morphological phenotypes, we utilized these transcriptional phenotypes to identify genes that are important for development by looking for transcripts whose abundance was unperturbed in most of the mutants. We found a set of 668 genes that includes many validated D. discoideum developmental genes. We have also found that abcG6 and abcG18 may have potential roles in intercellular signaling during terminal differentiation of spores and stalks. Transcriptional phenotyping during development of abc transporter mutants in Dictyostelium discoideum
Project description:Small RNAs play crucial roles in regulation of gene expression in many eukaryotes. Here, we report the cloning and characterization of 18-26 nt RNAs in the social amoeba Dictyostelium discoideum. This survey uncovered developmentally regulated microRNA candidates whose biogenesis, at least in one case, is dependent on a Dicer homolog, DrnB. Furthermore, we identified a large number of 21 nt RNAs originating from the DIRS-1 retrotransposon, clusters of which have been suggested to constitute centromeres. Small RNAs from another retrotransposon, Skipper, were significantly up-regulated in strains depleted of the second Dicer-like protein, DrnA, and a putative RNA-dependent RNA polymerase, RrpC. In contrast, the expression of DIRS-1 small RNAs was not altered in any of the analyzed strains. This suggests the presence of multiple RNAi pathways in D. discoideum. In addition, we isolated several small RNAs with antisense complementarity to mRNAs. Three of these mRNAs are developmentally regulated. Interestingly, all three corresponding genes express longer antisense RNAs from which the small RNAs may originate. In at least one case, the longer antisense RNA is complementary to the spliced but not the unspliced pre-mRNA, indicating synthesis by an RNA-dependent RNA polymerase. Keywords: cDNA library; small RNA sequencing The aim of this study was to investigate the small RNA (18-26 nt) profile of Dictyostelium discoideum during growth and development. For this reason, we cloned and sequenced pooled small RNAs from growing single cells and from two different multicellular stages (16 and 24 hours of development). cDNA libraries of 18-26 nt D. discoideum RNAs were constructed according to two different protocols (Lee, R.C. and Ambros, V. (2001) Science, 294; Lau, N.C. et al (2001) Science, 294). Briefly, total RNA was isolated from growing D. discoideum AX4 strain cells as well as from cells developed for 16 hours and 24 hours, and the fractions were subsequently pooled. After size fractionation and ligation of a 3’ linker, the RNA was divided into two fractions, one of which was directly ligated to a 5’ linker, thus selecting for small RNAs with 5’ monophosphates. Following RT-PCR, the PCR fragments were cloned and sequenced. The second fraction was subjected to reverse transcription directly after 3’ ligation. Apart from synthesizing a complementary DNA strand, the reverse transcriptase adds a few non-templated C residues at the 3’ end. These C:s were then hybridized to a DNA oligo with three 3’ G:s followed by RT-PCR, cloning and sequencing. This approach is insensitive of the nature of the 5’ end of the small RNA.
Project description:The social amoeba Dictyostelium discoideum is vulnerable to infection by the pathogen that causes Legionnaire's Disease, Legionella pneumophila. Dictystelium cells lacking the dual-specificity phospahatase DupA are at least partially resistant to infection, and strikingly the expression profile of uninfected dupA null cells mirrors the profile of infected wild-type cells to a considerable extent. This suggests that DupA has a key role in regulating host defence systems.
Project description:ATP-binding cassette (ABC) transporters can translocate a broad spectrum of molecules across the cell membrane including physiological cargo and toxins. ABC transporters are known for the role they play in resistance towards anticancer agents in chemotherapy of cancer patients. There are 68 ABC transporters annotated in the genome of the social amoeba Dictyostelium discoideum. We have characterized more than half of these ABC transporters through a systematic study of mutations in their genes. We have analyzed morphological and transcriptional phenotypes for these mutants during growth and development and found that most of the mutants exhibited rather subtle phenotypes. A few of the genes may share physiological functions, as reflected in their transcriptional phenotypes. Since most of the abc-transporter mutants showed subtle morphological phenotypes, we utilized these transcriptional phenotypes to identify genes that are important for development by looking for transcripts whose abundance was unperturbed in most of the mutants. We found a set of 668 genes that includes many validated D. discoideum developmental genes. We have also found that abcG6 and abcG18 may have potential roles in intercellular signaling during terminal differentiation of spores and stalks.
Project description:This project investigated the effect of Cln3-deficiency on protein secretion in the social amoeba Dictyostelium discoideum by performing LC-MS/MS on conditioned media harvested from starved WT and cln3- cells.
Project description:Micro (mi)RNAs regulate gene expression in many eukaryotic organisms where they control diverse biological processes. Their biogenesis, from primary transcripts to mature miRNAs, have been extensively characterized in animals and plants, showing distinct differences between these phylogenetically distant groups of organisms. However, little is known about miRNA biogenesis in organisms whose evolutionary position is placed in between plants and animals and/or in unicellular organisms. Here, we investigate miRNA maturation in the unicellular amoeba Dictyostelium discoideum, belonging to Amoebozoa, which branched out after plants but before animals. High-throughput sequencing of small RNAs and poly(A)-selected RNAs demonstrated that the Dicer-like protein DrnB is required, and essentially specific, for global miRNA maturation in D. discoideum. Our RNA-seq data also showed that longer miRNA transcripts accumulate in a drnB knock-out strain, generally preceded by a T-rich putative promoter motif. For two model miRNAs we defined the transcriptional start sites (TSSs) of primary (pri)-miRNAs and showed that they carry the RNA polymerase II specific m7G-cap. Different biogenesis pathways generate the 3’ ends of these pri-miRNAs, where pri-mir-1177 reads into the downstream gene, while pri-mir-1176 has a distinct non-polyadenylated end. This 3´-end is processed to shorter intermediates, stabilized in DrnB-depleted cells, of which some carry a short oligo(A)-tail. Furthermore, we identified 10 new miRNAs, all DrnB dependent and developmentally regulated. Thus, the miRNA machinery in D. discoideum shares features with both plants and animals, which is in agreement with its evolutionary position and perhaps also an adaptation to its complex lifestyle: unicellular growth and multicellular development.
Project description:There is a paucity of data regarding what proteins localize to the mitochondria in the social amoeba Dictyostelium discoideum. Therefore, here, we utilized high-throughput tandem mass tag (TMT)-based protein quantitation to identify proteins in the whole-cell lysate, as well as in crude and purified mitochondrial samples. We then calculated the ratio of a protein’s abundance in the mitochondria isolates, both crude and highly purified, versus its abundance in the whole-cell lysate. The resulting value, indicating its enrichment in mitochondrial preparations, was further normalized to the average enrichment ratio of 47 reference mitochondrial proteins, to compute the relative enrichment ratio. Using this approach, we identified 908 putative mitochondrial proteins. We ultimately combined proteomic and in silico analyses to yield a comprehensive mitochondrial protein compendium. Our compendium lays the foundation for future studies to understand the functions of conserved mitochondrial proteins in health and diseases using D. discoideum as the model. It also provides an entry to study many fascinating mitochondrial processes that are unique in protists. Additionally, through comparative genomics, our compendium will complement mitochondrial protein discovery in other organisms and may shed light on the evolution of mitochondrial proteomes and processes.