The Physarum polycephalum Genome Reveals Extensive Use of Prokaryotic Two-Component and Metazoan-Type Tyrosine Kinase Signaling

Physarum polycephalum is a well-studied microbial eukaryote with unique experimental attributes relative to other experimental model organisms. It has a sophisticated life cycle with several distinct stages including amoebal, flagellated, and plasmodial cells. It is unusual in switching between open and closed mitosis according to specific life-cycle stages. Here we present the analysis of the genome of this enigmatic and important model organism and compare it with closely related species. The genome is littered with simple and complex repeats and the coding regions are frequently interrupted by introns with a mean size of 100 bases. Complemented with extensive transcriptome data, we define approximately 31,000 gene loci, providing unexpected insights into earlyeukaryoteevolution.Wedescribeextensiveuseofhistidinekinase-basedtwo-componentsystemsandtyrosinekinasesignaling, the presence of bacterial and plant type photoreceptors (phytochromes, cryptochrome, and phototropin) and of plant-type pentatricopeptide repeat proteins, as well as metabolic pathways, and a cell cycle control system typically found in more complex eukaryotes. Our analysis characterizes P. polycephalum as a prototypical eukaryote with features attributed to the last common ancestor of Amorphea, that is, the Amoebozoa and Opisthokonts. Specifically, the presence of tyrosine kinases inAcanthamoeba and Physarum as representatives of two distantly related subdivisions ofAmoebozoa argues against the later emergence of tyrosine kinase signaling in the opisthokont lineage and also against the acquisition by horizontal gene transfe

Computational screen for spliceosomal RNA genes aids in defining the phylogenetic distribution of major and minor spliceosomal components

The RNA molecules of the spliceosome are critical for specificity and catalysis during splicing of eukaryotic pre-mRNA. In order to examine the evolution and phylogenetic distribution of these RNAs, we analyzed 149 eukaryotic genomes representing a broad range of phylogenetic groups. RNAs were predicted using high-sensitivity local alignment methods and profile HMMs in combination with covariance models. The results provide the most comprehensive view so far of the phylogenetic distribution of spliceosomal RNAs. RNAs were predicted in many phylogenetic groups where these RNA were not previously reported. Examples are RNAs of the major (U2-type) spliceosome in all fungal lineages, in lower metazoa and many protozoa. We also identified the minor (U12-type) spliceosomal U11 and U6atac RNAs in Acanthamoeba castellanii, where U12 spliceosomal RNA as well as minor introns were reported recently. In addition, minor-spliceosome-specific RNAs were identified in a number of phylogenetic groups where previously such RNAs were not observed, including the nematode Trichinella spiralis, the slime mold Physarum polycephalum and the fungal lineages Zygomycota and Chytridiomycota. The detailed map of the distribution of the U12-type RNA genes supports an early origin of the minor spliceosome and points to a number of occasions during evolution where it was lost

Intricate RNA : RNA Interactions in U12-Dependent Nuclear Pre-mRNA Splicing

Coding regions or exons of most human genes are interrupted by noncoding intervening regions or introns. Removal of nuclear precursor messenger RNA (pre-mRNA) introns by RNA splicing is an essential step in eukaryotic gene expression. Two types of nuclear pre-mRNA introns are known as U2-dependent or major type and U12-dependent or minor type. Nuclear pre-mRNA introns are removed by two distinct sets of ribonucleoprotein complexes or spliceosomes, which are formed by five small nuclear RNAs (snRNAs) for each spliceosome. U6atac and U12 snRNAs are central to U12-dependent spliceosome and play essential roles in the removal of U12-dependent introns. U6atac and U12 snRNAs bind to the 5\u27 splice site and branch site, respectively of an U12-dependent intron. In addition, it has been predicted that, U6atac and U12 snRNAs interact inter-molecularly to form helix I structure, which appears to be an essential element of the minor spliceosome. We have been studying U6atac and U12 inter-molecular base-pairing interaction using an in vivo mutation suppression assay. In this study, we have characterized U6atac and U12 mediated helix I intermolecular interactions and have shown in vivo existence of the predicted structure. In addition, we have also identified a region of U6atac snRNA which appears to be a structural analog of U12 snRNA stem III element. This element is important for the function of U12 snRNA and functions by binding to a RNA binding 65K protein, which is unique to minor spliceosome. We show that, analogous stem-loop of U6atac snRNA also interacts with 65K - RNA binding protein. However, functional significance of this interaction remained unclear. In summation, we have characterized sequential and dynamic RNA-RNA interactions between U4atac-U6atac and U6atac-U12 snRNAs. Our data show that, extensive and obligatory RNA-RNA interactions are critical to the splicing of U12-dependent intron

Functionally important structural elements of U12 snRNA

U12 snRNA is analogous to U2 snRNA of the U2-dependent spliceosome and is essential for the splicing of U12-dependent introns in metazoan cells. The essential region of U12 snRNA, which base pairs to the branch site of minor class introns is well characterized. However, other regions which are outside of the branch site base pairing region are not yet characterized and the requirement of these structures in U12-dependent splicing is not clear. U12 snRNA is predicted to form an intricate secondary structure containing several stem–loops and single-stranded regions. Using a previously characterized branch site genetic suppression assay, we generated second-site mutations in the suppressor U12 snRNA to investigate the in vivo requirement of structural elements in U12-dependent splicing. Our results show that stem–loop IIa is essential and required for in vivo splicing. Interestingly, an evolutionarily conserved stem–loop IIb is dispensable for splicing. We also show that stem–loop III, which binds to a p65 RNA binding protein of the U11-U12 di.snRNP complex, is essential for in vivo splicing. The data validate the existence of proposed stem–loops of U12 snRNA and provide experimental support for individual secondary structures

Unusual features of fibrillarin cDNA and gene structure in Euglena gracilis: evolutionary conservation of core proteins and structural predictions for methylation-guide box C/D snoRNPs throughout the domain Eucarya

Box C/D ribonucleoprotein (RNP) particles mediate O(2′)-methylation of rRNA and other cellular RNA species. In higher eukaryotic taxa, these RNPs are more complex than their archaeal counterparts, containing four core protein components (Snu13p, Nop56p, Nop58p and fibrillarin) compared with three in Archaea. This increase in complexity raises questions about the evolutionary emergence of the eukaryote-specific proteins and structural conservation in these RNPs throughout the eukaryotic domain. In protists, the primarily unicellular organisms comprising the bulk of eukaryotic diversity, the protein composition of box C/D RNPs has not yet been extensively explored. This study describes the complete gene, cDNA and protein sequences of the fibrillarin homolog from the protozoon Euglena gracilis, the first such information to be obtained for a nucleolus-localized protein in this organism. The E.gracilis fibrillarin gene contains a mixture of intron types exhibiting markedly different sizes. In contrast to most other E.gracilis mRNAs characterized to date, the fibrillarin mRNA lacks a spliced leader (SL) sequence. The predicted fibrillarin protein sequence itself is unusual in that it contains a glycine-lysine (GK)-rich domain at its N-terminus rather than the glycine-arginine-rich (GAR) domain found in most other eukaryotic fibrillarins. In an evolutionarily diverse collection of protists that includes E.gracilis, we have also identified putative homologs of the other core protein components of box C/D RNPs, thereby providing evidence that the protein composition seen in the higher eukaryotic complexes was established very early in eukaryotic cell evolution

The linked units of 5S rDNA and U1 snDNA of razor shells (Mollusca: Bivalvia: Pharidae)

[Abstract] The linkage between 5S ribosomal DNA and other multigene families has been detected in many eukaryote lineages, but whether it provides any selective advantage remains unclear. In this work, we report the occurrence of linked units of 5S ribosomal DNA (5S rDNA) and U1 small nuclear DNA (U1 snDNA) in 10 razor shell species (Mollusca: Bivalvia: Pharidae) from four different genera. We obtained several clones containing partial or complete repeats of both multigene families in which both types of genes displayed the same orientation. We provide a comprehensive collection of razor shell 5S rDNA clones, both with linked and nonlinked organisation, and the first bivalve U1 snDNA sequences. We predicted the secondary structures and characterised the upstream and downstream conserved elements, including a region at −25 nucleotides from both 5S rDNA and U1 snDNA transcription start sites. The analysis of 5S rDNA showed that some nontranscribed spacers (NTSs) are more closely related to NTSs from other species (and genera) than to NTSs from the species they were retrieved from, suggesting birth-and-death evolution and ancestral polymorphism. Nucleotide conservation within the functional regions suggests the involvement of purifying selection, unequal crossing-overs and gene conversions. Taking into account this and other studies, we discuss the possible mechanisms by which both multigene families could have become linked in the Pharidae lineage. The reason why 5S rDNA is often found linked to other multigene families seems to be the result of stochastic processes within genomes in which its high copy number is determinan

Ancient roles of non-coding RNAs in eukaryotic evolution

RNAs not coding for proteins, non-coding RNAs (ncRNAs) have many important roles in all kingdoms of life. Especially in eukaryotes, the regulatory functions of ncRNAs have been suggested as a major force in the evolution of complex traits. Cellular processes that are regulated by ncRNAs include for example cell differentiation, organ development and defense against viruses and transposable elements. This is achieved through a number of mechanisms like RNA destabilization and modification, transcriptional and translational control and chromatin modifications. Dictyostelium discoideum is a social amoeba and the best studied organism representing Amoebozoa, one of the eukaryotic supergroups. It has for long served as an excellent model for many basic cellular events like chemotaxis, differentiation and development and recently also for infection. The ncRNA population in D. discoideum is in many ways typical of eukaryotes but also harbors particularities. In this thesis I have studied spliceosomal RNAs as well as the RNA interference and microRNA pathways, which probably were present in the last eukaryotic common ancestor. I have also characterized Class I RNAs which seems to be specific to social amoebae. In addition, we have described the signal recognition particle RNA in several protists and also the involvement of a ncRNA during host interaction and stress in Giardia lamblia. Combining the well established molecular tools and knowledge about various pathways in D. discoideum, with the growing understanding of ncRNA, could in the future give important information about the function of ncRNAs as well as their ancient roles and evolution