RNA silencing is a form of post-transcriptional gene regulation, in which a small RNA guides a member of the Argonaute protein family in an effector complex to repress target gene expression. piRNAs, found in germ cells, are the most recently discovered major subset of small RNAs. A key known function of piRNA is to repress the transposable elements in the germline and maintain the germline genome integrity. The defining features of the piRNAs are 1) they are ubiquitously methylated at the 3’-end of the 2’-OH group by methyltransferase Hen1; 2) they associate exclusively with the Piwi subfamily Argonaute proteins. Much is not understood about the biogenesis and the regulation of the piRNA pathway. One of the fundamental questions is how the 3’-end of the piRNA is generated and recognised specifically by Piwi but not by Ago subfamily Argonaute proteins. In this thesis, the high resolution crystal structure of the Aubergine PAZ domain, a domain from a Piwi subfamily Argonaute, bound to a 7 mer single-stranded methylated piRNA ‘mimic’, reveals the mode of recognition for the 3’-end of piRNAs by Piwi subfamily Argonautes. The structure provides the molecular basis for why Piwi but not Ago PAZ domains preferentially bind to RNAs with 2’-O-methylation at the 3’-end, thus conferring substrate specificity. The structural results are confirmed by biochemical studies. Biochemical and biophysical studies on the methyltransferase Hen1 have provided insights into substrate specificity for piRNA 3’-end methylation and revealed a potential regulatory role for the C-terminal region of the protein. Extensive biochemical analysis defined a minimal active Hen1/short RNA complex, though crystallisation screening yielded no crystals for structure determination. Overall, this study provides insights into the generation and molecular recognition of the piRNA in animals.

Thesis (Ph. D.)--University of Missouri-Columbia, 2001. / Typescript. Vita. Includes bibliographical references. Also available on the Internet.

Thesis (Ph. D.)--University of Missouri-Columbia, 2001. / Typescript. Vita. Includes bibliographical references. Also available on the Internet.

The RFAM database defines families of ncRNAs by means of sequence similarities that are sufficient to establish homology. In some cases, such as microRNAs and box H/ACA snoRNAs, functional commonalities define classes of RNAs that are characterized by structural similarities, and typically consist of multiple RNA families. Recent advances in high-throughput transcriptomics and comparative genomics have produced very large sets of putative noncoding RNAs and regulatory RNA signals. For many of them, evidence for stabilizing selection acting on their secondary structures has been derived, and at least approximate models of their structures have been computed. The overwhelming majority of these hypothetical RNAs cannot be assigned to established families or classes. We present here a structure-based clustering approach that is capable of extracting putative RNA classes from genome-wide surveys for structured RNAs. The LocARNA (local alignment of RNA) tool implements a novel variant of the Sankoff algorithm that is sufficiently fast to deal with several thousand candidate sequences. The method is also robust against false positive predictions, i.e., a contamination of the input data with unstructured or nonconserved sequences. We have successfully tested the LocARNA-based clustering approach on the sequences of the RFAM-seed alignments. Furthermore, we have applied it to a previously published set of 3,332 predicted structured elements in the Ciona intestinalis genome (Missal K, Rose D, Stadler PF (2005) Noncoding RNAs in Ciona intestinalis. Bioinformatics 21 (Supplement 2): i77–i78). In addition to recovering, e.g., tRNAs as a structure-based class, the method identifies several RNA families, including microRNA and snoRNA candidates, and suggests several novel classes of ncRNAs for which to date no representative has been experimentally characterized.

We report an efficient method for detecting functional RNAs. The approach, which combines comparative sequence analysis and structure prediction, already has yielded excellent results for a small number of aligned sequences and is suitable for large-scale genomic screens. It consists of two basic components: (i) a measure for RNA secondary structure conservation based on computing a consensus secondary structure, and (ii) a measure for thermodynamic stability, which, in the spirit of a z score, is normalized with respect to both sequence length and base composition but can be calculated without sampling from shuffled sequences. Functional RNA secondary structures can be identified in multiple sequence alignments with high sensitivity and high specificity. We demonstrate that this approach is not only much more accurate than previous methods but also significantly faster. The method is implemented in the program rnaz, which can be downloaded from www.tbi.univie.ac.at/~wash/RNAz. We screened all alignments of length n ≥ 50 in the Comparative Regulatory Genomics database, which compiles conserved noncoding elements in upstream regions of orthologous genes from human, mouse, rat, Fugu, and zebrafish. We recovered all of the known noncoding RNAs and cis-acting elements with high significance and found compelling evidence for many other conserved RNA secondary structures not described so far to our knowledge.

If has been proposed that a spontaneous deletion in the nicotinamide nucleotide transhydrogenase (Nnt) gene eliminating exons 7-11 in C57BL/6J (B6J) mice is associated with reduced glucose-stimulated insulin secretion in vitro, impaired glucose tolerance, higher epigonadal fat mass and altered susceptibility to diet induced obesity (DIO) of male B6J mice. A potential implication for NNT in human adipose tissue distribution has not been investigated so far. We therefore analyzed NNT mRNA expression in paired human samples of visceral (vis) and subcutaneous (sc) adipose tissue from 221 subjects with a wide range of BMI, insulin sensitivity and glucose tolerance. NNT mRNA expression is significantly higher in visceral fat of obese patients and correlates with body weight, BMI, % body fat, visceral and sc fat area, waist and hip circumference as well fasting plasma insulin. Multivariate linear regression analysis revealed visceral fat area, and % body fat, but not fasting plasma insulin and 2h OGTT glucose. In conclusion, our data suggest a functional relevance of NNT in the development of human obesity and visceral fat distribution.

In contrast to the fairly reliable and complete annotation of the protein coding genes in the human genome, comparable information is lacking for noncoding RNAs (ncRNAs). We present a comparative screen of vertebrate genomes for structural noncoding RNAs, which evaluates conserved genomic DNA sequences for signatures of structural conservation of base-pairing patterns and exceptional thermodynamic stability. We predict more than 30,000 structured RNA elements in the human genome, almost 1,000 of which are conserved across all vertebrates. Roughly a third are found in introns of known genes, a sixth are potential regulatory elements in untranslated regions of protein-coding mRNAs and about half are located far away from any known gene. Only a small fraction of these sequences has been described previously. A comparison with recent tiling array data shows that more than 40% of the predicted structured RNAs overlap with experimentally detected sites of transcription. The widespread conservation of secondary structure points to a large number of functional ncRNAs and cis-acting mRNA structures in the human genome.