The how and why of lncRNA function: An innate immune perspective.

Next-generation sequencing has provided a more complete picture of the composition of the human transcriptome indicating that much of the "blueprint" is a vastness of poorly understood non-protein-coding transcripts. This includes a newly identified class of genes called long noncoding RNAs (lncRNAs). The lack of sequence conservation for lncRNAs across species meant that their biological importance was initially met with some skepticism. LncRNAs mediate their functions through interactions with proteins, RNA, DNA, or a combination of these. Their functions can often be dictated by their localization, sequence, and/or secondary structure. Here we provide a review of the approaches typically adopted to study the complexity of these genes with an emphasis on recent discoveries within the innate immune field. Finally, we discuss the challenges, as well as the emergence of new technologies that will continue to move this field forward and provide greater insight into the biological importance of this class of genes. This article is part of a Special Issue entitled: ncRNA in control of gene expression edited by Kotb Abdelmohsen

Systems biology in inflammatory bowel diseases

Purpose of review: Ulcerative colitis (UC) and Crohn’s Disease (CD) are the two predominant types of inflammatory bowel disease (IBD), affecting over 1.4 million individuals in the US. IBD results from complex interactions between pathogenic components, including genetic and epigenetic factors, the immune response and the microbiome through an unknown sequence of events. The purpose of this review is to describe a system biology approach to IBD as a novel and exciting methodology aiming at developing novel IBD therapeutics based on the integration of molecular and cellular "omics" data. Recent Findings: Recent evidence suggested the presence of genetic, epigenetic, transcriptomic, proteomic and metabolomic alterations in IBD patients. Furthermore, several studies have shown that different cell types, including fibroblasts, epithelial, immune and endothelial cells together with the intestinal microbiota are involved in IBD pathogenesis. Novel computational methodologies have been developed aiming to integrate high - throughput molecular data. Summary: A systems biology approach could potentially identify the central regulators (hubs) in the IBD interactome and improve our understanding of the molecular mechanisms involved in IBD pathogenesis. The future IBD therapeutics should be developed on the basis of targeting the central hubs in the IBD network

CLIP and complementary methods

RNA molecules start assembling into ribonucleoprotein (RNP) complexes during transcription. Dynamic RNP assembly, largely directed by cis-acting elements on the RNA, coordinates all processes in which the RNA is involved. To identify the sites bound by a specific RNA-binding protein on endogenous RNAs, cross-linking and immunoprecipitation (CLIP) and complementary, proximity-based methods have been developed. In this Primer, we discuss the main variants of these protein-centric methods and the strategies for their optimization and quality assessment, as well as RNA-centric methods that identify the protein partners of a specific RNA. We summarize the main challenges of computational CLIP data analysis, how to handle various sources of background and how to identify functionally relevant binding regions. We outline the various applications of CLIP and available databases for data sharing. We discuss the prospect of integrating data obtained by CLIP with complementary methods to gain a comprehensive view of RNP assembly and remodelling, unravel the spatial and temporal dynamics of RNPs in specific cell types and subcellular compartments and understand how defects in RNPs can lead to disease. Finally, we present open questions in the field and give directions for further development and applications

Terrae Incognitae: Integrative Genomic Analysis of Hnrnp L Splicing Regulation

Alternative splicing is a critical component of human gene control that generates functional diversity from a limited genome. Defects in alternative splicing are associated with disease in humans. Alternative splicing is regulated developmentally and physiologically by the combinatorial actions of cis- and trans-acting factors, including RNA binding proteins that regulate splicing through sequence-specific interactions with pre-mRNAs. In T cells, the splicing regulator hnRNP L is an essential factor that regulates alternative splicing of physiologically important mRNAs, however the broader physical and functional impact of hnRNP L remains unknown. In this dissertation, I present analysis of hnRNP L-RNA interactions with CLIP-seq, which identifies transcriptome-wide binding sites and uncovers novel functional targets. I then use functional genomics studies to define pre-mRNA processing alterations induced by hnRNP L depletion, chief among which is cassette-type alternative splicing. Finally, I use integrative genomic analysis to identify a direct role for hnRNP L in repression of exon inclusion and an indirect role for enhancing exon inclusion that supports a novel regulatory interplay between hnRNP L and chromatin. In two appendices, I present CLIP-seq studies of two additional RNA binding proteins: the splicing factor CELF2 and the RNA helicase DDX17. In conclusion, I provide comparisons of these three CLIP-seq studies, providing high-level insights into the capabilities and limitations of CLIP-seq. In sum, this dissertation expands our knowledge of hnRNP L splicing control in the context of broader studies of RNA binding proteins in multiple cell types and conditions

Fox-1 family of RNA-binding proteins

The Fox-1 family of RNA-binding proteins are evolutionarily conserved regulators of tissue-specific alternative splicing in metazoans. The Fox-1 family specifically recognizes the (U)GCAUG stretch in regulated exons or in flanking introns, and either promotes or represses target exons. Recent unbiased bioinformatics analyses of alternatively spliced exons and comparison of various vertebrate genomes identified the (U)GCAUG stretch as a highly conserved and widely distributed element enriched in intronic regions surrounding exons with altered inclusion in muscle, heart, and brain, consistent with specific expression of Fox-1 and Fox-2 in these tissues. Global identification of Fox-2 target RNAs in living cells revealed that many of the Fox-2 target genes themselves encode splicing regulators. Further systematic elucidation of target genes of the Fox-1 family and other splicing regulators in various tissues will lead to a comprehensive understanding of splicing regulatory networks

Genome-Wide Decoding of mRNP and miRNA Maps

The limited number of primary transcripts in the genome has promoted interest in the possibility that much of the complexity in the regulation of gene expression may be determined by RNA regulation controlled by RNA-binding proteins (RNABPs) and/or microRNAs (miRNAs). However, applying biochemical methods to understand such interactions in living tissues is major challenge. Here we developed a genome-wide means of mapping messenger ribonucleoprotein (mRNP) sites in vivo, by high-throughput sequencing of RNA isolated by crosslinking immunoprecipitation (HITS-CLIP). HITS-CLIP analysis of the neuron-specific splicing factor Nova provides genome-wide maps of Nova-RNA interactions in vivo and leads to a new finding that Nova may regulate the processesing of some miRNAs. Furthermore, HITS-CLIP analysis is extended to the problem of identifying miRNA targets, for which prediction is a major challenge since miRNA activity requires base pairing through only 6-8 “seed†nucleotides. By generating crosslinking of native Argonaute (Ago) protein-RNA complexes in mouse brain, Ago HITS-CLIP produced two simultaneous datasets—Ago-miRNA and Ago-mRNA binding sites—that were combined with bioinformatic analysis to identify miRNA-target mRNA interaction sites. We validated genome-wide interaction maps for miR-124, and generated additional maps for the 20 most abundant miRNAs present in P13 mouse brain. We also found that the relatively large number of Ago proteins bind in coding sequence, as well as introns, suggesting unexplored functions for miRNAs. Not all Ago mRNA clusters correspond to known seed sequence, leading to the discovery of putative new rules for miRNA-mRNA interactions. HITS-CLIP provides a general plaform to identify functional mRNP and miRNA binding sites in vivo and a solution to determining precise sequences for targeting clinically relevant sites of RNA regulation. In addition, overlaying mRNP maps with miRNA maps will be informative for the understanding of RNA regulations and complexity