 My name is David Stanik and I'm the head of the robot of RNA biology at the Institute of Morcology and Ethics in Prague, the Czech Republic. And our research is oriented on RNA splicing and diseases connected to RNA splicing. And one of these diseases is so-called retinosis pigmentosa. Retinosis pigmentosa is a hereditary disease which affects one out of four thousand individuals. And during the disease the retinasos are dying, people are losing the night vision, they lose the peripheral vision, going to so-called the tunnel vision and eventually they can be totally blind. Most mutated genes that cause this disease are somehow connected with the function of photoreceptor cells or developmental of retina. But surprisingly five genes which are mutated and causing retinosis pigmentosa were connected to primary splicing and formation of splicing machinery. And all these genes are part of the core spliceosomal particles called small ribonucleoprotein particles or SNURPs. And five genes connected to the disease were HPRP8, HPRP6, HPRP31, HPRP3 and human birth 2. And mutation in all these particles were studied and all causes problems in assembly of the core spliceosomal particle SNURPs. However the initial research only suggested that mutations in birth 2 doesn't affect SNURP assembly and that's why we decided to study birth 2 mutations in human cells. And that was the task for my postdoc Suska Cvacikova. Hello, I'm Susana Cvacikova, I'm postdoc in this lab. We studied two mutations of BRR2. They both results in amino acid alteration and are situated in N-terminal helicase domain of BRR2 that plays an important role in RNA duplex unwinding. BRR2 is a large protein of 200 kilodaltons and it would be quite difficult to prepare its mutants by traditional side-directed mutagenesis. We overcame this problem and employed BRR2 that is expressed from bacterial artificial chromosome and that is tagged with GFP. We prepared both mutants by Bach recombinering technique. Using of Bach provides additional advantages. It preserves endogenous promoter and other regulatory sequences, exon intrastructure and our protein is expressed at near physiological level. We were also able to knock down specifically endogenous BRR2 using a CRNA designed against the sequence close to the stop codon where GFP type is attached. Wild type and both mutants were stably expressed in helicels. They both reveal a similar speckled distribution pattern and they both colloquialized with SRSF2 which is a marker of nuclear speckles. As David told, retinitis pigmentosa linked mutations of splicing factor compromise trisnerp assembly. We tested incorporation of BRR2 mutants into snrps by immunoprecipitation using antibody and we observed that both mutants were properly incorporated in snrps as you can see for example on this RNA gel. To test the role of BRR2 mutants on splicing, we studied alternative splicing of some endogenous genes and gene reporters based on genes that are abundantly expressed in the retina and there was no difference between wild type and mutants. But then we observed that BRR2 mutants increase efficiency of constitutive splicing. So we decided to have a look on how precisely do BRR2 mutants their job. We employed beta-globin based reporters containing cryptic splice site. Cryptic splice site is much more used than the normal splice site is mutated. We studied splicing of beta-globin reporters after knockdown of endogenous BRR2. In Hela cells this resulted in less efficient splicing of wild type beta-globin reporter and enhanced splicing of mutated beta-globin reporter. Expression of either BRR2 mutant did not reduce splicing efficiency of mutated beta-globin reporter. Spicing efficiency was similar as in BRR2 depleted cells. It is pigmentosa linked mutations, vegan proof reading function of BRR2 and this could be a serious problem for metabolically highly active cells such as photoreceptors. I would like to thank you for your attention. The whole story was recently published in Journal of Human Mutation and if you are interested you can read it there and I wish you good reading. Thank you.