No description available.

No description available.

No description available.

No description available.

No description available.

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.

No description available.

This is the first of 2 review papers on genetics and genomics appearing as part of the series on “omics.” Genomics pertains to all components of an organism’s genes, whereas genetics involves analysis of a specific gene(s) in the context of heredity. The paper provides introductory comments, describes the basis of human genetic diversity, and addresses the phenotypic consequences of genetic variants. Rare variants with large effect sizes are responsible for single gene disorders, whereas complex polygenic diseases are typically due to multiple genetic variants, each exerting a modest effect size. To illustrate the clinical implications of genetic variants with large effect sizes, 3 common forms of hereditary cardiomyopathies are discussed as prototypic examples of single-gene disorders, including their genetics, clinical manifestations, pathogenesis, and treatment. The genetic basis of complex traits is discussed in a separate paper.

It was shown that 95 % of human multi-exon genes are alternatively spliced and the regulation of alternative splicing is extremely complex. Most pre-mRNA splicing events occur co- transcriptionally and there is increasing body of evidence, that chromatin modifications play an important role in the regulation of alternative splicing. Here we showed that inhibition of histone deacetylases (HDACs) modulates alternative splicing of ~700 genes via induction of histone H4 acetylation and increase of Pol II elongation rate along alternative region. We identified HDAC1 the catalytic activity of which is responsible for changes in alternative splicing. Then, we analyzed whether acetylhistone binding protein Brd2 regulates alternative splicing and showed that Brd2 occupies promoter regions of targeted genes and controls alternative splicing of ~300 genes. Later we showed that knockdown of histone acetyltransferase p300 promotes inclusion of the alternative fibronectin (FN1) EDB exon. p300 associates with CRE sites in the promoter via the CREB transcription factor. We created mini-gene reporters driven by an artificial promoter containing CRE sites. Both deletion and mutation of the CRE site affected EDB alternative splicing in the same manner as the p300 knockdown. Next we showed that p300 controls histone...

A new series of fluorescent prorin (promazine-coumarin hybrid) derivatives were synthesized via piperidine catalyzed cyclization with 3-formyl-2-hydroxypromazine and ethyl acetoacetate or Meldrum’s acid. The synthesis of these new compounds focused on the introduction of electron withdrawing carbonyl groups on the 3-position of the coumarin lactone ring in order to increase polarization of the molecule which results in a longer absorbance wavelength by stabilizing intramolecular charge transfer that occurs in the excited state. The 3-acetyl derivative was further subjected to an aldol condensation with either benzaldehyde or cinnamaldehyde in order to further extend the conjugation of the molecule. The four new prorin compounds exhibited excellent photophysical properties and had Stokes shifts ranging from 160-190 nm. These properties are ideal for use as fluorescent probes to detect RNA binding ligands. The optical properties of the new prorin compounds were compared with a previous series of prorins as well as theoretical calculations in order to gain a more comprehensive understanding of how the photophysical properties of the molecule can be tuned by introducing different substituents. Although increasing polarization of the molecule with electron withdrawing substituents does red-shift the absorbance wavelength, it has little effect on the emission wavelength and can actually cause a blue-shift in some cases. It was observed that introducing aromatic substituents has the greatest effect on red-shifting the emission wavelength and increasing Stokes shift.