Martin Kiefmann

RNomics: an experimental approach that identifies 201 candidates for novel, small, non‐messenger RNAs in mouse

In mouse brain cDNA libraries generated from small RNA molecules we have identified a total of 201 different expressed RNA sequences potentially encoding novel small non‐messenger RNA species (snmRNAs). Based on sequence and structural motifs, 113 of these RNAs can be assigned to the C/D box or H/ACA box subclass of small nucleolar RNAs (snoRNAs), known as guide RNAs for rRNA. While 30 RNAs represent mouse homologues of previously identified human C/D or H/ACA snoRNAs, 83 correspond to entirely novel snoRNAs. Among these, for the first time, we identified four C/D box snoRNAs and four H/ACA box snoRNAs predicted to direct modifications within U2, U4 or U6 small nuclear RNAs (snRNAs). Furthermore, 25 snoRNAs from either class lacked antisense elements for rRNAs or snRNAs. Therefore, additional snoRNA targets have to be considered. Surprisingly, six C/D box snoRNAs and one H/ACA box snoRNA were expressed exclusively in brain. Of the 88 RNAs not belonging to either snoRNA subclass, at least 26 are probably derived from truncated heterogeneous nuclear RNAs (hnRNAs) or mRNAs. Short interspersed repetitive elements (SINEs) are located on five RNA sequences and may represent rare examples of transcribed SINEs. The remaining RNA species could not as yet be assigned either to any snmRNA class or to a part of a larger hnRNA/mRNA. It is likely that at least some of the latter will represent novel, unclassified snmRNAs.

Retroposed elements as archives for the evolutionary history of placental mammals.

Reconstruction of the placental mammalian (eutherian) evolutionary tree has undergone diverse revisions, and numerous aspects remain hotly debated. Initial hierarchical divisions based on morphology contained many misgroupings due to features that evolved independently by similar selection processes. Molecular analyses corrected many of these misgroupings and the superordinal hierarchy of placental mammals was recently assembled into four clades. However, long or rapid evolutionary periods, as well as directional mutation pressure, can produce molecular homoplasies, similar characteristics lacking common ancestors. Retroposed elements, by contrast, integrate randomly into genomes with negligible probabilities of the same element integrating independently into orthologous positions in different species. Thus, presence/absence analyses of these elements are a superior strategy for molecular systematics. By computationally scanning more than 160,000 chromosomal loci and judiciously selecting from only phylogenetically informative retroposons for experimental high-throughput PCR applications, we recovered 28 clear, independent monophyly markers that conclusively verify the earliest divergences in placental mammalian evolution. Using tests that take into account ancestral polymorphisms, multiple long interspersed elements and long terminal repeat element insertions provide highly significant evidence for the monophyletic clades Boreotheria (synonymous with Boreoeutheria), Supraprimates (synonymous with Euarchontoglires), and Laurasiatheria. More importantly, two retropositions provide new support for a prior scenario of early mammalian evolution that places the basal placental divergence between Xenarthra and Epitheria, the latter comprising all remaining placentals. Due to its virtually homoplasy-free nature, the analysis of retroposon presence/absence patterns avoids the pitfalls of other molecular methodologies and provides a rapid, unequivocal means for revealing the evolutionary history of organisms.

Experimental RNomics: Identification of 140 Candidates for Small Non-Messenger RNAs in the Plant Arabidopsis thaliana

BACKGROUND Genomes from all organisms known to date express two types of RNA molecules: messenger RNAs (mRNAs), which are translated into proteins, and non-messenger RNAs, which function at the RNA level and do not serve as templates for translation. RESULTS We have generated a specialized cDNA library from Arabidopsis thaliana to investigate the population of small non-messenger RNAs (snmRNAs) sized 50-500 nt in a plant. From this library, we identified 140 candidates for novel snmRNAs and investigated their expression, abundance, and developmental regulation. Based on conserved sequence and structure motifs, 104 snmRNA species can be assigned to novel members of known classes of RNAs (designated Class I snmRNAs), namely, small nucleolar RNAs (snoRNAs), 7SL RNA, U snRNAs, as well as a tRNA-like RNA. For the first time, 39 novel members of H/ACA box snoRNAs could be identified in a plant species. Of the remaining 36 snmRNA candidates (designated Class II snmRNAs), no sequence or structure motifs were present that would enable an assignment to a known class of RNAs. These RNAs were classified based on their location on the A. thaliana genome. From these, 29 snmRNA species located to intergenic regions, 3 located to intronic sequences of protein coding genes, and 4 snmRNA candidates were derived from annotated open reading frames. Surprisingly, 15 of the Class II snmRNA candidates were shown to be tissue-specifically expressed, while 12 are encoded by the mitochondrial or chloroplast genome. CONCLUSIONS Our study has identified 140 novel candidates for small non-messenger RNA species in the plant A. thaliana and thereby sets the stage for their functional analysis.

Mesozoic retroposons reveal parrots as the closest living relatives of passerine birds

The relationships of passerines (such as the well-studied zebra finch) with non-passerine birds is one of the great enigmas of avian phylogenetic research, because decades of extensive morphological and molecular studies yielded highly inconsistent results between and within data sets. Here we show the first application of the virtually homoplasy-free retroposon insertions to this controversy. Our study examined ~200,000 retroposon-containing loci from various avian genomes and retrieved 51 markers resolving early bird phylogeny. Among these, we obtained statistically significant evidence that parrots are the closest and falcons the second-closest relatives of passerines, together constituting the Psittacopasserae and the Eufalconimorphae, respectively. Our new and robust phylogenetic framework has substantial implications for the interpretation of various conclusions drawn from passerines as model organisms. This includes insights of relevance to human neuroscience, as vocal learning (that is, birdsong) probably evolved in the psittacopasseran ancestor, >30 million years earlier than previously assumed.

Evolution of small nucleolar RNAs in nematodes

In contrast to mRNAs, which are templates for translating proteins, non-protein coding (npc) RNAs (also known as ‘non-coding’ RNA, ncRNA), exhibit various functions in different compartments and developmental stages of the cell. Small nucleolar RNAs (snoRNAs), one of the largest classes of npcRNAs, guide post-transcriptional modifications of other RNAs that are crucial for appropriate RNA folding as well as for RNA–RNA and RNA–protein interactions. Although snoRNA genes comprise a significant fraction of the eutherian genome, identifying and characterizing large numbers of them is not sufficiently accessible by classical computer searches alone. Furthermore, most previous investigations of snoRNAs yielded only limited indications of their evolution. Using data obtained by a combination of high-throughput cDNA library screening and computational search strategies based on a modified DNAMAN program, we characterized 151 npcRNAs, and in particular 121 snoRNAs, from Caenorhabditis elegans and extensively compared them with those in the related, Caenorhabditis briggsae. Detailed comparisons of paralog snoRNAs in the two nematodes revealed, in addition to trans-duplication, a novel, cis-duplication distribution strategy with insertions near to the original loci. Some snoRNAs coevolved with their modification target sites, demonstrating the close interaction of complementary regions. Some target sites modified by snoRNAs were changed, added or lost, documenting a high degree of evolutionary plasticity of npcRNAs.

Genome sequence of the basal haplorrhine primate Tarsius syrichta reveals unusual insertions.

Tarsiers are phylogenetically located between the most basal strepsirrhines and the most derived anthropoid primates. While they share morphological features with both groups, they also possess uncommon primate characteristics, rendering their evolutionary history somewhat obscure. To investigate the molecular basis of such attributes, we present here a new genome assembly of the Philippine tarsier (Tarsius syrichta), and provide extended analyses of the genome and detailed history of transposable element insertion events. We describe the silencing of Alu monomers on the lineage leading to anthropoids, and recognize an unexpected abundance of long terminal repeat-derived and LINE1-mobilized transposed elements (Tarsius interspersed elements; TINEs). For the first time in mammals, we identify a complete mitochondrial genome insertion within the nuclear genome, then reveal tarsier-specific, positive gene selection and posit population size changes over time. The genomic resources and analyses presented here will aid efforts to more fully understand the ancient characteristics of primate genomes.