Gene expression evidence for off-target effects caused by RNA interference-mediated gene silencing of Ubiquitin-63E in the cattle tick Rhipicephalus microplus

Knowledge of cattle tick (Rhipicephalus (Boophilus) microplus; Acari: Ixodidae) molecular and cellular pathways has been hampered by the lack of an annotated genome. In addition, most of the tick expressed sequence tags (ESTs) available to date consist of not, vert, similar50% unassigned sequences without predicted functions. The most common approach to address this has been the application of RNA interference (RNAi) methods to investigate genes and their pathways. This approach has been widely adopted in tick research despite minimal knowledge of the tick RNAi pathway and double-stranded RNA (dsRNA) uptake mechanisms. A strong knockdown phenotype of adult female ticks had previously been observed using a 594 bp dsRNA targeting the cattle tick homologue for the Drosophila Ubiquitin-63E gene leading to nil or deformed eggs. A NimbleGen cattle tick custom microarray based on the BmiGI.V2 database of R. microplus ESTs was used to evaluate the expression of mRNAs harvested from ticks treated with the tick Ubiquitin-63E 594 bp dsRNA compared with controls. A total of 144 ESTs including TC6372 (Ubiquitin-63E) were down-regulated with 136 ESTs up-regulated following treatment. The results obtained substantiated the knockdown phenotype with ESTs identified as being associated with ubiquitin proteolysis as well as oogenesis, embryogenesis, fatty acid synthesis and stress responses. A bioinformatics analysis was undertaken to predict off-target effects (OTE) resulting from the in silico dicing of the 594 bp Ubiquitin-63E dsRNA which identified 10 down-regulated ESTs (including TC6372) within the list of differentially expressed probes on the microarrays. Subsequent knockdown experiments utilising 196 and 109 bp dsRNAs, and a cocktail of short hairpin RNAs (shRNA) targeting Ubiquitin-63E, demonstrated similar phenotypes for the dsRNAs but nil effect following shRNA treatment. Quantitative reverse transcriptase PCR analysis confirmed differential expression of TC6372 and selected ESTs. Our study demonstrated the minimisation of predicted OTEs in the shorter dsRNA treatments (not, vert, similar100–200 bp) and the usefulness of microarrays to study knockdown phenotypes

Optimizing property codes in protein data reveals structural characteristics

Abstract. We search for assignments of numbers to the amino acids (property codes) that maximize the autocorrelation function signal in given protein sequence data by an iterative method. Our method yields similar results to optimization with the related extended Jacobi method for joint diagonalization and standard optimization tools. In nonhomologous sets representative of all proteins we find optimal property codes that are similar to hydrophobicity but yield much clearer correlations. Another property code related to α-helix propensity plays a less prominent role representing a local optimum. We also apply our method to sets of proteins known to have a high content of α- or βstructures and find property codes reflecting the specific correlations in these structures