Insights into protein-RNA complexes from computational analyses of iCLIP experiments

RNA-binding proteins (RBPs) are the primary regulators of all aspects of posttranscriptional gene regulation. In order to understand how RBPs perform their function, it is important to identify their binding sites. Recently, new techniques have been developed to employ high-throughput sequencing to study protein-RNA interactions in vivo, including the individual-nucleotide resolution UV crosslinking and immunoprecipitation (iCLIP). iCLIP identifies sites of protein-RNA crosslinking with nucleotide resolution in a transcriptome-wide manner. It is composed of over60steps,whichcanbemodified,butitisnotclearhowvariationsinthemethod affect the assignment of RNA binding sites. This is even more pertinent given that several variants of iCLIP have been developed. A central question of my research is how to correctly assign binding sites to RBPs using the data produced by iCLIP and similar techniques. I first focused on the technical analyses and solutions for the iCLIP method. I examinedcDNAdeletionsandcrosslink-associatedmotifstoshowthatthestartsof cDNAs are appropriate to assign the crosslink sites in all variants of CLIP, including iCLIP, eCLIP and irCLIP. I also showed that the non-coinciding cDNA-starts are caused by technical conditions in the iCLIP protocol that may lead to sequence constraintsatcDNA-endsinthefinalcDNAlibrary. Ialsodemonstratedtheimportance of fully optimizing the RNase and purification conditions in iCLIP to avoid thesecDNA-endconstraints. Next,IdevelopedCLIPo,acomputationalframework that assesses various features of iCLIP data to provide quality control standards which reveals how technical variations between experiments affect the specificity of assigned binding sites. I used CLIPo to compare multiple PTBP1 experiments produced by iCLIP, eCLIP and irCLIP, to reveal major effects of sequence constraintsatcDNA-endsorstarts,cDNAlengthdistributionandnon-specificcontaminants. Moreover, I assessed how the variations between these methods influence themechanisticconclusions. Thus,CLIPopresentsthequalitycontrolstandardsfor transcriptome-wide assignment of protein-RNA binding sites. I continued with analyses of RBP complexes by using data from spliceosomeiCLIP. This method simultaneously detects crosslink sites of small nuclear ribonucleoproteins (snRNPs) and auxiliary splicing factors on pre-mRNAs. I demonstratedthatthehighresolutionofspliceosome-iCLIPallowsfordistinctionbetween multiple proximal RNA binding sites, which can be valuable for transcriptomewide studies of large ribonucleoprotein complexes. Moreover, I showed that spliceosome-iCLIP can experimentally identify over 50,000 human branch points. In summary, I detected technical biases from iCLIP data, and demonstrated how such biases can be avoided, so that cDNA-starts appropriately assign the RNA binding sites. CLIPo analysis proved a useful quality control tool that evaluates data specificity across different methods, and I applied it to iCLIP, irCLIP and ENCODE eCLIP datasets. I presented how spliceosome-iCLIP data can be used to study the splicing machinery on pre-mRNAs and how to use constrained cDNAs from spliceosome-iCLIP data to identify branch points on a genome-wide scale. Taken together, these studies provide new insights into the field of RNA biology and can be used for future studies of iCLIP and related methods

Existence and Uniqueness of Tri-tronqu\'ee Solutions of the second Painlev\'e hierarchy

The first five classical Painlev\'e equations are known to have solutions described by divergent asymptotic power series near infinity. Here we prove that such solutions also exist for the infinite hierarchy of equations associated with the second Painlev\'e equation. Moreover we prove that these are unique in certain sectors near infinity.Comment: 13 pages, Late

Autoresonance in a Dissipative System

We study the autoresonant solution of Duffing's equation in the presence of dissipation. This solution is proved to be an attracting set. We evaluate the maximal amplitude of the autoresonant solution and the time of transition from autoresonant growth of the amplitude to the mode of fast oscillations. Analytical results are illustrated by numerical simulations.Comment: 22 pages, 3 figure

Approximations for quantiles of life expectancy and annuity values using the parametric improvement rate approach for modelling and projecting mortality

In this paper, we develop accurate approximations for medians of life expectancy and life annuity pure premiums viewed as functions of future mortality trends as predicted by parametric models of the improvement rates in mortality. Numerical illustrations show that the comonotonic approximations perform well in this case, which suggests that they can be used in practice to evaluate the consequences of the uncertainty in future death rates. Prediction intervals based on 5% and 95% quantiles are also considered but appear to be wider compared to simulated ones. This provides the practitioner with a conservative shortcut, thereby avoiding the problem of simulations within simulations in, for instance, Solvency 2 calculations

Data Science Issues in Understanding Protein-RNA Interactions

An interplay of experimental and computational methods is required to achieve a comprehensive understanding of protein–RNA interactions. UV crosslinking and immunoprecipitation (CLIP) identifies endogenous interactions by sequencing RNA fragments that copurify with a selected RNA-binding protein under stringent conditions. Here we focus on approaches for the analysis of the resulting data and appraise the methods for peak calling, visualization, analysis, and computational modeling of protein–RNA binding sites. We advocate that the sensitivity and specificity of data be assessed in combination for computational quality control. Moreover, we demonstrate the value of analyzing sequence motif enrichment in peaks assigned from CLIP data and of visualizing RNA maps, which examine the positional distribution of peaks around regulated landmarks in transcripts. We use these to assess how variations in CLIP data quality and in different peak calling methods affect the insights into regulatory mechanisms. We conclude by discussing future opportunities for the computational analysis of protein–RNA interaction experiments

Psoriasis Vulgaris: A Genetic Approach

Evidence for a genetic contribution in psoriasis comes from direct examination of a large segment of the population in an isolated island environment, epidemiologic and questionnaire studies presented to psoriatic patients, twin studies collected from the literature and from twin registries, and splitsibship analysis. The concordance of psoriasis in monozygotic twins was 65–72%, whereas psoriasis in dizygotic twins was 15–30%. Determination of concordance in older twin pairs from a national twin registry in Denmark revealed nearly 90–100% heritability. In order to link psoriasis with known markers within the human genome, serologic studies have been carried out with a variety of blood group and polymorphic protein antigens. A weak association with the MNS and Lewis Blood Groups Systems (relative risk, 3.5) has been identified. Stronger associations with class I B locus and class II D locus genes (relative risk, 8–12) have also been determined by studies of the human lymphocyte-antigen system. Finally, a strong association with HLA Cw6 has been determined; this marker is thought to be in linkage disequilibrium with B and D locus genes previously associated with psoriasis. The relative risk of developing psoriasis in HLA Cw6 positive individuals is about 24. A few large kindred have been reported in the dermatology literature. These support the hypothesis of autosomal dominant inheritance with penetrance of approximately 60%. In cooperation with The National Psoriasis Foundation, we have now identified over 90 families with psoriasis in three generations. We have begun the process of ascertainment, the construction of family trees, and the collection of leukocyte DNA for linkage analysis with established restriction fragment polymorphisms (RFLP). Our initial assessment is being directed to four RFLP that span approximately 30 centiMorgans of the short arm of human chromosome 6. Although karyotyping is uncommoly done in patients because of psoriasis, we now seek evidence of translocation of chromosome 6 in association with psoriasis