17-704 Sigma-AldrichMagna RIP™ Quad RNA-Binding Protein Immunoprecipitation Kit

MicroRNAs (miRNAs) are generally recognized as regulating gene expression posttranscriptionally by inhibiting translation or inducing target mRNA degradation. New mechanisms for miRNAs to regulate gene expression also still attract much attention. More and more novel miRNAs are discovered by the advanced sequencing technology, but yet their biological functions are largely unknown. Up to now, the function of miR-466l, a miRNA discovered in mouse embryonic stem cells, remains unclear. In this study, we report that miR-466l can upregulate both mRNA and protein expression of IL-10 in TLR-triggered macrophages. Furthermore, we show that miR-466l can competitively bind to the IL-10 3' untranslated region AU-rich elements, which is a typical binding site for RNA-binding protein (RBP). Tristetraprolin is a well-known RBP, and mediates rapid degradation of IL-10 mRNA. miRNA always mediates target mRNA degradation or translation repression modestly; thus, the net effect of miR-466l's binding to IL-10 AU-rich elements is to prevent IL-10 mRNA degradation mediated by tristetraprolin, resulting in extended t(1/2) of IL-10 mRNA and elevated IL-10 expression. Thus, competitive binding with RBP to the same target mRNA and subsequent stabilization of target mRNA is an alternative mechanism for gene regulation by miRNAs. Also, a mechanism for regulation of IL-10 by miRNAs is outlined.

In eukaryotic organisms, gene regulatory networks require an additional level of coordination that links transcriptional and post-transcriptional processes. Messenger RNAs have traditionally been viewed as passive molecules in the pathway from transcription to translation. However, it is now clear that RNA-binding proteins (RBPs) play a major role in regulating multiple mRNAs to facilitate gene expression patterns. On this basis, post-transcriptional and transcriptional gene expression networks appear to be very analogous. Our previous research focused on targeting RBPs to develop a better understanding of post-transcriptional gene-expression processing and the regulation of mRNA networks. We developed technologies for purifying endogenously formed RBP-mRNA complexes from cellular extracts and identifying the associated messages using genome-scale, microarray technology, a method called ribonomics or RNA-binding protein immunoprecipitation-microarray (Chip) profiling or RIP-Chip. The use of the RIP-Chip methods has provided great insight into the infrastructure of coordinated eukaryotic post-transcriptional gene expression, insights which could not have been obtained using traditional RNA expression profiling approaches (1). This chapter describes the most current RIP-Chip techniques as we presently practice them. We also discuss some of the informatic aspects that are unique to analyzing RIP-Chip data.

In this chapter, we present an approach using genomic and ribonomic profiling to investigate functional gene programs in a tumor growth model. To reach this goal, ribonomic profiling was combined with RNA interference in a tumor dormancy model. Strategies merging functional genomic technologies are outlined for the identification of novel posttranscriptionally regulated targets of p38 to show that they are functionally linked to the induction or interruption of cellular growth in cancer. In the first section of this chapter, we describe a method for the detection of mRNA subsets associated with RNA-binding proteins such as hnRNP A1 using (1) immunopurification of mRNA-protein complexes, from either whole cell lysates or subcellular fractions and (2) gene expression arrays to find those mRNAs bound to hnRNP A1. In the second section, short hairpin RNA technology was used to create a library of shRNAs that target p38 induced mRNAs expression libraries are utilized to "knockdown" the genes identified in the first section. Finally, this library of gene candidates is evaluated in vivo to address their functional role in the induction or maintenance of dormancy.

RNA targets of multitargeted RNA-binding proteins (RBPs) can be studied by various methods including mobility shift assays, iterative in vitro selection techniques and computational approaches. These techniques, however, cannot be used to identify the cellular context within which mRNAs associate, nor can they be used to elucidate the dynamic composition of RNAs in ribonucleoprotein (RNP) complexes in response to physiological stimuli. But by combining biochemical and genomics procedures to isolate and identify RNAs associated with RNA-binding proteins, information regarding RNA-protein and RNA-RNA interactions can be examined more directly within a cellular context. Several protocols--including the yeast three-hybrid system and immunoprecipitations that use physical or chemical cross-linking--have been developed to address this issue. Cross-linking procedures in general, however, are limited by inefficiency and sequence biases. The approach outlined here, termed RNP immunoprecipitation-microarray (RIP-Chip), allows the identification of discrete subsets of RNAs associated with multi-targeted RNA-binding proteins and provides information regarding changes in the intracellular composition of mRNPs in response to physical, chemical or developmental inducements of living systems. Thus, RIP-Chip can be used to identify subsets of RNAs that have related functions and are potentially co-regulated, as well as proteins that are associated with them in RNP complexes. Using RIP-Chip, the identification and/or quantification of RNAs in RNP complexes can be accomplished within a few hours or days depending on the RNA detection method used.

RNA-binding proteins can organize messenger RNAs (mRNAs) into structurally and functionally related subsets, thus facilitating the coordinate production of gene classes necessary for complex cellular processes. Historically, in vitro methods primarily have been used to identify individual targets of mRNA-binding proteins. However, more direct methods are required for the identification of endogenously associated RNAs and their cognate proteins. To better understand posttranscriptional mRNA organization within the cell, we developed a systems biology approach to identify multiple-endogenous mRNA transcripts associated with RNA-binding proteins. This approach, termed ribonomics, takes advantage of high-throughput genomic array technologies that have greatly advanced the study of global gene expression changes. This chapter describes techniques for purifying mRNA-protein complexes (mRNPs) and identifying the associated mRNAs

Although in vitro methods have been used to identify putative targets of mRNA-binding proteins, direct in vivo methods are needed to identify endogenously associated mRNAs and their cognate proteins. Therefore, we have developed high-throughput methods to identify structurally and/or functionally related mRNA transcripts through their endogenous association with RNA-binding proteins. We have termed the identification and analysis of mRNA subsets using RNA-associated proteins ribonomics, and have established four primary steps for the method: (1) isolation of endogenous mRNA-protein complexes (mRNPs) under optimized conditions, (2) the en masse characterization of the protein and mRNA components associated with the targeted mRNP complexes, (3) identification of sequences or structural similarities among members of the mRNA subset, and (4) determination of functional relationships among the protein products coded for by members of the mRNA subset. We have hypothesized that mRNAs are organized into structurally and functionally linked groups to better affect information transfer through coordinate gene expression. The functional consequences of such organization would be to facilitate the production of proteins that regulate processes necessary for growth and differentiation. This article describes a series of biochemical techniques that deal with the first two steps of ribonomic profiling: purifying endogenous mRNP complexes and identifying multiple mRNA targets using microarray analysis.

Genomic array technologies provide a means for profiling global changes in gene expression under a variety of conditions. However, it has been difficult to assess whether transcriptional or posttranscriptional regulation is responsible for these changes. Additionally, fluctuations in gene expression in a single cell type within a complex tissue like a tumor may be masked by overlapping profiles of all cell types in the population. In this paper, we describe the use of cDNA arrays to identify subsets of mRNAs contained in endogenous messenger ribonucleoprotein complexes (mRNPs) that are cell type specific. We identified mRNA subsets from P19 embryonal carcinoma stem cells by using mRNA-binding proteins HuB, eIF-4E, and PABP that are known to play a role in translation. The mRNA profiles associated with each of these mRNPs were unique and represented gene clusters that differed from total cellular RNA. Additionally, the composition of mRNAs detected in HuB-mRNP complexes changed dramatically after induction of neuronal differentiation with retinoic acid. We suggest that the association of structurally related mRNAs into mRNP complexes is dynamic and may help regulate posttranscriptional events such as mRNA turnover and translation. Recovering proteins specifically associated with mRNP complexes to identify and profile endogenously clustered mRNAs should provide insight into structural and functional relationships among gene transcripts and/or their protein products. We have termed this approach to functional genomics ribonomics and suggest that it will provide a useful paradigm for organizing genomic information in a biologically relevant manner.