Characterization of post-transcriptional regulatory network of RNA-binding proteins using computational predictions and deep sequencing data

This report is divided into three parts: Data Analysis, Mathematical Modeling and Conclusion and future directions. In the Data Analysis part, various methods and tools for characterizing the post-transcriptional regulatory networks of RNA-binding proteins are discussed and applied. Chapter 2 introduces PAR-CLIP, a method for transcriptomewide identification of RNA binding proteins at nucleotide resolution. PAR-CLIP was successfully applied on RNA binding proteins and their binding specificity was characterized. Partly due to their vast volume, the data that were so far generated in CLIP experiments have not been put in a form that enables fast and interactive exploration of binding sites. To address this need, Chapter 3 presents CLIPZ, which is a database and analysis environment for various kinds of deep sequencing (and in particular CLIP) data, that aims to provide an open-access repository of information for post-transcriptional regulatory elements. Chapter 4 revisits various CLIP methods. A set of ideas in terms of both experimental protocols and data analysis are presented to improve the quality and reproducibility of such experiments. In general, cytoplasmic RNAs are isolated in CLIP experiments. Like many high-throughput experiments, CLIP has a certain amount of isolated RNAs which do not represent regulatory binding sites. To improve the quality of the obtained RNAs, a set of novel methods for data analysis are also suggested. These methods are added as new tools to the CLIPZ analysis platform. Argonaute CLIP data could in principle be beneficial in improving the microRNA target site predictions. However, several questions still remain which cannot be addressed using CLIP methods. For example: • Argonaute CLIP data by default does not reveal which microRNAs are more likely to interact to the mRNA binding site at the time of cross-linking. • As mentioned earlier, biochemical and structural studies of Thermus thermophilus Argonaute protein suggest that the protein-RNA interaction between microRNA and the Argonaute protein forms a physical structure that only some positions in the microRNA become accessible to the target binding site. Having inferred the interacting microRNA, it is also interesting to predict the most plausible secondary structure of the hybridized microRNA-mRNA complex. Mathematical Modeling part of the report contains Chapter 5. This chapter presents a novel mathematical model called MIRZA to address the above mentioned questions. An in-depth introduction to MIRZA is presented and its performance in terms of identifying functionally relevant targets of microRNAs is discussed. Finally, Conclusion and future directions part of the report contains Chapter 6 in which discusses the main findings of the projects and gives an outlook of where future work could be taken up

CLIPZ: a database and analysis environment for experimentally determined binding sites of RNA-binding proteins

The stability, localization and translation rate of mRNAs are regulated by a multitude of RNA-binding proteins (RBPs) that find their targets directly or with the help of guide RNAs. Among the experimental methods for mapping RBP binding sites, cross-linking and immunoprecipitation (CLIP) coupled with deep sequencing provides transcriptome-wide coverage as well as high resolution. However, partly due to their vast volume, the data that were so far generated in CLIP experiments have not been put in a form that enables fast and interactive exploration of binding sites. To address this need, we have developed the CLIPZ database and analysis environment. Binding site data for RBPs such as Argonaute 1-4, Insulin-like growth factor II mRNA-binding protein 1-3, TNRC6 proteins A-C, Pumilio 2, Quaking and Polypyrimidine tract binding protein can be visualized at the level of the genome and of individual transcripts. Individual users can upload their own sequence data sets while being able to limit the access to these data to specific users, and analyses of the public and private data sets can be performed interactively. CLIPZ, available at http://www.clipz.unibas.ch, aims to provide an open access repository of information for post-transcriptional regulatory element

Conserved generation of short products at piRNA loci

ABSTRACT: BACKGROUND: The piRNA pathway operates in animal germ lines to ensure genome integrity through retrotransposon silencing. The Piwi protein-associated small RNAs (piRNAs) guide Piwi proteins to retrotransposon transcripts, which are degraded and thereby post-transcriptionally silenced through a ping-pong amplification process. Cleavage of the retrotransposon transcript defines at the same time the 5` end of a secondary piRNA that will in turn guide a Piwi protein to a primary piRNA precursor, thereby amplifying primary piRNAs. Although several studies provided evidence that this mechanism is conserved among metazoa, how the process is initiated and what enzymatic activities are responsible for generating the primary and secondary piRNAs are not entirely clear. RESULTS: Here we analyzed small RNAs from three mammalian species, seeking to gain further insight into the mechanisms responsible for the piRNA amplification loop. We found that in all these species piRNA-directed targeting is accompanied by the generation of short sequences that have a very precisely defined length, 19 nucleotides, and a specific spatial relationship with the guide piRNAs. CONCLUSIONS: This suggests that the processing of the 5` product of piRNA-guided cleavage occurs while the piRNA target is engaged by the Piwi protein. Although they are not stabilized through methylation of their 3` ends, the 19-mers are abundant not only in testes lysates but also in immunoprecipitates of Miwi and Mili proteins. They will enable more accurate identification of piRNA loci in deep sequencing data sets

The study of effectiveness of Schema Therapy in Reducing the Activity of the Early Maladaptive Schema in Women with Social Anxiety Disorder

The purpose of this study was to determine the effectiveness of schema therapy in decreasing the maladaptive schemas of women with social anxiety disorder. The research method was a single-case experimental design with multiple baselines. Five patients were selected using purpose sampling method. They attended in 20 sessions of therapy individually according to Young's schema therapy model. The instruments used in this study were tests of social anxiety and Young Schema Questionnaire (YSQ: long form). The improved diagnostic method and clinical significance were used for analyzing the data. The results showed that Young's schema therapy model reduced the activity of the early maladaptive schemas significantly in all areas of schemas. The results of follow-up showed the treatment had been persistent and effective. The Young's schema therapy model can be used for the treatment of chronic mental disorders that early maladaptive schemas play a primarily role in their persistance

Original Article 128 128 An Association Study on IL16 Gene Polymorphisms with the Risk of Sporadic Alzheimer's Disease

Abstract Background: is an important regulator of T cell activation and was reported to act as a chemoattractant agent. There are evidences that IL16 can control the neuroinflammatory processes in Alzheimer's Disease (AD). This study was performed to investigate the role or association of IL16 polymorphisms, rs11556218 and rs4778889 with the risk of late-onset Alzheimer's disease (LOAD) in Iranian population. Methods: Totally, 148 AD patients and 137 nondemented and age-matched subjects were recruited in this study. Genotyping of rs11556218 T/G and rs4778889 T/C polymorphisms was performed by PCR-RFLP method using the NdeI and AhdI restriction enzymes, respectively. Results: Statistical analysis of rs11556218 genotypes showed a protective effect against AD in the heterozygote genotype (p=0.001, OR=0.16) as well as rs4778889 (p=0.001, OR=0.23). Frequency of rs11556218 allele T was higher in controls than patients (p= 0.001, OR=0.32). However, there was no significant difference in the frequencies of rs4778889 alleles between the AD patients and controls. Conclusion: Our results indicate that the rs11556218 and rs4778889 polymorphisms have a protective role in the development of sporadic AD in Iranian population

A quantitative analysis of CLIP methods for identifying binding sites of RNA-binding proteins

Cross-linking and immunoprecipitation (CLIP) is increasingly used to map transcriptome-wide binding sites of RNA-binding proteins. We developed a method for CLIP data analysis, and applied it to compare CLIP with photoactivatable ribonucleoside-enhanced CLIP (PAR-CLIP) and to uncover how differences in cross-linking and ribonuclease digestion affect the identified sites. We found only small differences in accuracies of these methods in identifying binding sites of HuR, which binds low-complexity sequences, and Argonaute 2, which has a complex binding specificity. We found that cross-link-induced mutations led to single-nucleotide resolution for both PAR-CLIP and CLIP. Our results confirm the expectation from original CLIP publications that RNA-binding proteins do not protect their binding sites sufficiently under the denaturing conditions used during the CLIP procedure, and we show that extensive digestion with sequence-specific RNases strongly biases the recovered binding sites. This bias can be substantially reduced by milder nuclease digestion conditions