Web-based tools for studying RNA structure and function.

Like protein coding sequences, functional motifs in RNA elements are frequently conserved, but this conservation is most often at the structure level rather than sequence based. Proper characterization of these structural RNA motifs is both the key and the limiting step to understanding the nature of RNA-protein interactions. The discovery of elements targeted by RNA-binding proteins and how they function remains one of the most active, yet elusive areas of RNA biology. Only a limited number of these elements have been well characterized with many of the fundamental rules yet to be discovered. Here we present a comprehensive list of web based resources that can be used in the study and identification of RNA-based structural and regulatory motifs and provide a survey of the informatic resources that can have been developed to facilitate this research.

RIP-Chip analysis: RNA-Binding Protein Immunoprecipitation-Microarray (Chip) Profiling.

Post-transcriptional regulation of gene expression plays an important role in complex cellular processes. Just like transcription factors regulate gene expression through combinatorial binding to multiple, physically dispersed cis elements, mRNA binding proteins can regulate the translation of functionally related gene products by coordinately binding to subsets of mRNAs. The networks of mRNA binding proteins that facilitate this fine-tuning of gene expression are poorly understood. By combining genomic technologies with standard molecular biology tools, we have helped pioneer the development of high-throughput technologies for the global analysis of subsets of mRNAs bound to RNA-binding proteins. This technique is termed RIP-Chip and stands for RNA-Binding Protein Immunoprecipitation-Microarray (Chip) Profiling. This approach is also referred to as "ribonomic profiling" and has revealed valuable information about the workings of mRNP networks in the cell and the regulation of gene expression. In this chapter, we describe the latest advances that we have made in the RIP-CHIP technology.

RIP-CHIP in drug development.

Microarrays are extensively used to evaluate the effects of compounds on gene expression in the cells. Most of the studies so far have analyzed the transcriptome of the cell. The basic assumption of this approach is that the changes in gene expression occur at the level of transcription of a gene. However, changes often occur at the posttranscriptional level and are not reflected in the analysis of whole transcriptome. We have pioneered the development of "ribonomic profiling" as a high-throughput method to study posttranscriptional regulation of gene expression in the cell. This method is also often referred to as RIP-CHIP. In this chapter, we describe how to use the RIP-CHIP technology to assess the posttranscriptional changes occurring in the cell in response to treatment with a drug.

Informatic resources for identifying and annotating structural RNA motifs.

Post-transcriptional regulation of genes and transcripts is a vital aspect of cellular processes, and unlike transcriptional regulation, remains a largely unexplored domain. One of the most obvious and most important questions to explore is the discovery of functional RNA elements. Many RNA elements have been characterized to date ranging from cis-regulatory motifs within mRNAs to large families of non-coding RNAs. Like protein coding genes, the functional motifs of these RNA elements are highly conserved, but unlike protein coding genes, it is most often the structure and not the sequence that is conserved. Proper characterization of these structural RNA motifs is both the key and the limiting step to understanding the post-transcriptional aspects of the genomic world. Here, we focus on the task of structural motif discovery and provide a survey of the informatics resources geared towards this task.

Systems based mapping demonstrates that recovery from alkylation damage requires DNA repair, RNA processing, and translation associated networks.

The identification of cellular responses to damage can promote mechanistic insight into stress signalling. We have screened a library of 3968 Escherichia coli gene-deletion mutants to identify 99 gene products that modulate the toxicity of the alkylating agent methyl methanesulfonate (MMS). We have developed an ontology mapping approach to identify functional categories over-represented with MMS-toxicity modulating proteins and demonstrate that, in addition to DNA re-synthesis (replication, recombination, and repair), proteins involved in mRNA processing and translation influence viability after MMS damage. We have also mapped our MMS-toxicity modulating proteins onto an E. coli protein interactome and identified a sub-network consisting of 32 proteins functioning in DNA repair, mRNA processing, and translation. Clustering coefficient analysis identified seven highly connected MMS-toxicity modulating proteins associated with translation and mRNA processing, with the high connectivity suggestive of a coordinated response. Corresponding results from reporter assays support the idea that the SOS response is influenced by activities associated with the mRNA-translation interface.

Bioinformatic tools for studying post-transcriptional gene regulation : The UAlbany TUTR collection and other informatic resources.

The untranslated regions (UTRs) of many mRNAs contain sequence and structural motifs that are used to regulate the stability, localization, and translatability of the mRNA. It should be possible to discover previously unidentified RNA regulatory motifs by examining many related nucleotide sequences, which are assumed to contain a common motif. This is a general practice for discovery of DNA-based sequence-based patterns, in which alignment tools are heavily exploited. However, because of the complexity of sequential and structural components of RNA-based motifs, simple-alignment tools are frequently inadequate. The consensus sequences they find frequently have the potential for significant variability at any given position and are only loosely characterized. The development of RNA-motif discovery tools that infer and integrate structural information into motif discovery is both necessary and expedient. Here, we provide a selected list of existing web-accessible algorithms for the discovery of RNA motifs, which, although not exhaustive, represents the current state of the art. To facilitate the development, evaluation, and training of new software programs that identify RNA motifs, we created the UAlbany training UTR (TUTR) database, which is a collection of validated sets of sequences containing experimentally defined regulatory motifs. Presently, eleven training sets have been generated with associated indexes and "answer sets" provided that identify where the previously characterized RNA motif [the iron responsive element (IRE), AU-rich class-2 element (ARE), selenocysteine insertion sequence (SECIS), etc.] resides in each sequence. The UAlbany TUTR collection is a shared resource that is available to researchers for software development and as a research aid.

Advances in RIP-chip analysis : RNA-binding protein immunoprecipitation-microarray profiling.

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.

Genome-wide analysis identifies interleukin-10 mRNA as target of tristetraprolin.

Tristetraprolin (TTP) is an RNA-binding protein required for the rapid degradation of mRNAs containing AU-rich elements. Targets regulated by TTP include the mRNAs encoding tumor necrosis factor-alpha, granulocyte-macrophage colony-stimulating factor, interleukin-2 (IL-2), and immediate early response 3. To identify novel target mRNAs of TTP in macrophages, we used a genome-wide approach that combines RNA immunoprecipitation and microarray analysis. A list was compiled of 137 mRNAs that are associated with TTP with an estimated accuracy on the order of 90%. Sequence analysis revealed a highly significant enrichment of AU-rich element motifs, with AUUUA pentamers present in 96% and UUAUUUAUU nonamers present in 44% of TTP-associated mRNAs. We further show that IL-10 is a novel target regulated by TTP. IL-10 mRNA levels were found to be elevated because of a reduced decay rate in primary macrophages from TTP(-/-) mice. Our study demonstrates the importance of experimental approaches for identifying targets of RNA-binding proteins.

Microarray analysis in B cells among siblings with/without MS - role for transcription factor TCF2.

BACKGROUND: We investigated if global gene expression and transcription networks in B-lymphocytes of siblings with multiple sclerosis (MS) were different from healthy siblings. RESULTS: Using virus-transformed immortalized B cells and human whole genome bioarrays with validation using RT-qPCR, we found that in siblings with MS, genes for CXCL10, serpin B1 and FUT4 were up regulated whereas CDC5L, TNFRSF19 and HLA-DR were down regulated, among others; transcription analysis showed two intersecting clusters of transcriptional factors - the larger, governed by the upregulated transcription factor 2 (TCF2) and the smaller network regulated by the downregulated CDC5L. CONCLUSION: No study has linked TCF2 to MS and to better understand the role of TCF2 in MS, studies in larger cohorts are required.

MicroRNA modulation of RNA-binding protein regulatory elements.

We propose that microRNAs could modulate RNA-binding protein binding sites in a dynamic manner. We suggest that the cis-regulatory code targeted by microRNAs is, at least in part, the same as that read by mRNA-binding proteins. Our hypothesis predicts that microRNAs indirectly or directly bind to RNA-binding protein binding sites. Alternatively, the microRNA-mRNA interactions can themselves be the target of an mRNA-binding protein. Lastly, we envision examples where multiple mRNA regulatory elements are simultaneously influenced by microRNA-mRNA interactions such that the binding of one or more microRNA results in conformational changes in the structure of the mRNA, thereby, either revealing or masking a second regulatory element.