Detection Of RNA-binding Proteins Research Articles

RBPPred: predicting RNA-binding proteins from sequence using SVM.

Detection of RNA-binding proteins (RBPs) is essential since the RNA-binding proteins play critical roles in post-transcriptional regulation and have diverse roles in various biological processes. Moreover, identifying RBPs by computational prediction is much more efficient than experimental methods and may have guiding significance on the experiment design. In this study, we present the RBPPred (an RNA-binding protein predictor), a new method based on the support vector machine, to predict whether a protein binds RNAs, based on a comprehensive feature representation. By integrating the physicochemical properties with the evolutionary information of protein sequences, the new approach RBPPred performed much better than state-of-the-art methods. The results show that RBPPred correctly predicted 83% of 2780 RBPs and 96% out of 7093 non-RBPs with MCC of 0.808 using the 10-fold cross validation. Furthermore, we achieved a sensitivity of 84%, specificity of 97% and MCC of 0.788 on the testing set of human proteome. In addition we tested the capability of RBPPred to identify new RBPs, which further confirmed the practicability and predictability of the method. RBPPred program can be accessed at: http://rnabinding.com/RBPPred.html . liushiyong@gmail.com. Supplementary data are available at Bioinformatics online.

Detection of RNA-binding Proteins by <em>In Vitro</em> RNA Pull-down in Adipocyte Culture

Detection of RNA-binding Proteins by <em>In Vitro</em> RNA Pull-down in Adipocyte Culture

Detection of RNA-binding Proteins by In Vitro RNA Pull-down in Adipocyte Culture.

RNA-binding proteins (RBPs) are emerging as a regulatory layer in the development and function of adipose. RBPs play a key role in the gene expression regulation at posttranscriptional levels by affecting the stability and translational efficiency of target mRNAs. RNA pull-down technique has been widely used to study RNA-protein interaction, which is necessary to elucidate the mechanism underlying RBPs' as well as long non-coding RNAs' (lncRNAs) function. However, the high lipid abundance in adipocytes poses a technical challenge in conducting this experiment. Here a detailed RNA pull-down protocol is optimized for primary adipocyte culture. An RNA fragment from androgen receptor's (AR) 3' untranslated region (3'UTR) containing an adenylate-uridylate-rich elementwas used as an example to demonstrate how to retrieve its RBP partner, HuR protein, from adipocyte lystate. The method described here can be applied to detect the interactions between RBPs and noncoding RNAs, as well as between RBPs and coding RNAs.

Visualization of RNA binding proteins by sequential gel shift and ultraviolet cross-linking

RNA binding proteins partially constitute theribonucleoprotein or protein machinery for RNA processing (splicing, polyadenylation and 3′ end formation), transport, and storage. We have devised a novel method for the detection of RNA binding proteins in vitro. The template for transcription is a cloned Drosophila melanogaster 5S rRNA gene. The method is a two-dimensional gel analysis involving: in vitro transcription of 32P-labeled 5S rRNA using a cellular S-100; resolution of labeled RNA protein complexes from unbound RNA on a first-dimension mobility shift gel; cross-linking of RNA to protein in gel by ultraviolet irradiation; degradation of the RNA by RNase A and Tl; and analysis of 32P-protein patterns on a second-dimension discontinuous SDS gel by autoradiography. The pattern of proteins associated with 32P-5S rRNA is obtained by covalent transfer of 32P-nucleotides from RNA to the proteins with which the RNA was bound. This method could be useful in the analysis of RNA maturation and processing pathways.