Mammalian Cis-Acting RNA Sequence Elements

Cis-acting regulatory sequence elements are sequences contained in the 3′ and 5′ untranslated region, introns, or coding regions of precursor RNAs and mature mRNAs that are selectively recognized by a complementary set of one or more trans-acting factors to regulate posttranscriptional gene expression. This chapter focuses on mammalian cis-acting regulatory elements that had been recently discovered in different regions: pre-processed and mature. The chapter begins with an overview of two large networks of mRNAs that contain conserved AU-rich elements (AREs) or GU-rich elements (GREs), and their role in mammalian cell physiology. Other, less conserved, cis-acting elements and their functional role in different steps of RNA maturation and metabolism will be discussed. The molecular characteristics of pathological cis-acting sequences that rose from gene mutations or transcriptional aberrations are briefly outlined, with the proposed approach to restore normal gene expression. Concise models of the function of posttranscriptional regulatory networks within different cellular compartments conclude this chapter

DSCAM-AS1-Driven Proliferation of Breast Cancer Cells Involves Regulation of Alternative Exon Splicing and 3'-End Usage.

DSCAM-AS1 is a cancer-related long noncoding RNA with higher expression levels in Luminal A, B, and HER2-positive Breast Carcinoma (BC), where its expression is strongly dependent on Estrogen Receptor Alpha (ERα). DSCAM-AS1 expression is analyzed in 30 public datasets and, additionally, by qRT-PCR in tumors from 93 BC patients, to uncover correlations with clinical data. Moreover, the effect of DSCAM-AS1 knockdown on gene expression and alternative splicing is studied by RNA-Seq in MCF-7 cells. We confirm DSCAM-AS1 overexpression in high grade Luminal A, B, and HER2+ BCs and find a significant correlation with disease relapse. In total, 908 genes are regulated by DSCAM-AS1-silencing, primarily involved in the cell cycle and inflammatory response. Noteworthily, the analysis of alternative splicing and isoform regulation reveals 2085 splicing events regulated by DSCAM-AS1, enriched in alternative polyadenylation sites, 3'UTR (untranslated region) shortening and exon skipping events. Finally, the DSCAM-AS1-interacting splicing factor heterogeneous nuclear ribonucleoprotein L (hnRNPL) is predicted as the most enriched RBP for exon skipping and 3'UTR events. The relevance of DSCAM-AS1 overexpression in BC is confirmed by clinical data and further enhanced by its possible involvement in the regulation of RNA processing, which is emerging as one of the most important dysfunctions in cancer

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

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

microRNAs of parasitic helminths – identification, characterization and potential as drug targets

microRNAs (miRNAs) are small non-coding RNAs involved in post-transcriptional gene regulation. They were first identified in the free-living nematode Caenorhabditis elegans, where the miRNAs lin-4 and let-7 were shown to be essential for regulating correct developmental progression. The sequence of let-7 was subsequently found to be conserved in higher organisms and changes in expression of let-7, as well as other miRNAs, are associated with certain cancers, indicating important regulatory roles. Some miRNAs have been shown to have essential functions, but the roles of many are currently unknown. With the increasing availability of genome sequence data, miRNAs have now been identified from a number of parasitic helminths, by deep sequencing of small RNA libraries and bioinformatic approaches. While some miRNAs are widely conserved in a range of organisms, others are helminth-specific and many are novel to each species. Here we review the potential roles of miRNAs in regulating helminth development, in interacting with the host environment and in development of drug resistance. Use of fluorescently-labeled small RNAs demonstrates uptake by parasites, at least in vitro. Therefore delivery of miRNA inhibitors or mimics has potential to alter miRNA activity, providing a useful tool for probing the roles of miRNAs and suggesting novel routes to therapeutics for parasite control

Purification of UV cross-linked RNA-protein complexes by phenol-toluol extraction

RNA-Bindungsproteine spielen Schlüsselfunktionen bei der post-transkriptionellen Regulation der Genexpression. Durch Bindung an RNA steuern sie die RNA-Aufbereitung, den Transport, die Stabilität und die Translation. In den letzten zehn Jahren wurden bedeutende Fortschritte bei der Aufklärung bakterieller post-transkriptioneller Mechanismen erzielt. Es wird immer deutlicher, dass diese Regulierungsebene auch bei der Pathogenese und Antibiotikaresistenz eine wichtige Rolle spielt. Die Analyse von RNA-Protein-Komplexen (RNPs) auf Proteomebene wurde durch die (m)RNA-interactome-capture Technologie vorangetrieben, die den Teil des Proteoms isoliert, welcher mit polyadenylierter (m)RNA vernetzt ist. Dies hat zur Identifizierung von Hunderten von neuen RBPs in einer Vielzahl von eukaryontischen Arten, vom Menschen bis zur Hefe, geführt. Allerdings fehlt die Poly-Adenylierung in der funktionellen RNA von Bakterien und anderen Klassen von -eukaryontischen- regulatorischen RNAs. Ziel dieser Arbeit war es, diese Einschränkung durch die Entwicklung einer neuartigen und unvoreingenommenen Methode zur Aufreinigung von UV-vernetzten RNPs in lebenden Zellen zu überwinden: PTex (Phenol-Toluol-Extraktion). Das Reinigungsprinzip basiert ausschließlich auf den physikalisch-chemischen Eigenschaften von vernetzten RNPs gegenüber ungebundenen Proteinen oder RNA; es ist dabei unparteiisch gegenüber spezifischen RNAs oder Proteinen und ermöglicht somit erstmals eine systemweite Analyse von nicht-poly-(A)-RNA-interagierenden Proteinen sowohl in eukaryontischen (HEK293) als auch in prokaryontischen (Salmonella Typhimurium) Zellen.RNA binding proteins play key functions in post-transcriptional regulation of gene expression. By binding to RNA, they control RNA editing, transport, stability and translation. In the last decade, significant advances have been made in the elucidation of bacterial post-transcriptional mechanisms. It is becoming increasingly clear that this layer of regulation also plays an important role in pathogenesis and antibiotic resistance. The analysis of RNA-protein complexes (RNPs) at the proteome level has been driven by the (m)RNA interactome capture technology which isolates the proteome cross-linked to poly-adenylated (m)RNA. This has resulted in the identification of hundreds of novel RBPs in a diversity of eukaryotic species ranging from humans to yeast. However, poly-adenylation is absent in functional RNA from bacteria and other classes of -eukaryotic- regulatory RNAs. This work was aimed to overcome that limitation by developing a novel and unbiased method for the purification of UV-cross-linked RNPs in living cells: PTex (Phenol Toluol extraction). The purification principle is solely based on physicochemical properties of cross-linked RNPs versus unbound proteins or RNA, and it is impartial towards specific RNA or proteins; enabling for the first time a system-wide analysis of non-poly(A) RNA interacting proteins in both eukaryotic (HEK293) and prokaryotic (Salmonella Typhimurium) cells

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

Characterizing Human Transfer RNAS by Hydro-TRNASEQ and PAR-CLIP

The participation of tRNAs in fundamental aspects of biology and disease necessitates an accurate, experimentally confirmed annotation of tRNA genes, and curation of precursor and mature tRNA sequences. This has been challenging, mainly because RNA secondary structure and nucleotide modifications, together with tRNA gene multiplicity, complicate sequencing and read mapping efforts. To address these issues, I developed hydro-tRNAseq, a method based on partial alkaline RNA hydrolysis that generates fragments amenable for sequencing. To identify transcribed tRNA genes, I further complemented this approach with Photoactivatable Crosslinking and Immunoprecipitation (PAR-CLIP) of SSB/La, a conserved protein involved in pre-tRNA processing. My results show that approximately half of all predicted tRNA genes are transcribed in human cells, suggesting that the tRNA genomic space is more contracted than previously thought as a result of regulation of expression. I also report predominant nucleotide modification sites, their order of incorporation, and identify tRNA leader, trailer and intron sequences. By using complementary sequencing-based methodologies I present a human tRNA reference set, and determine expression levels of mature and processing intermediates of tRNAs in human cells. The technical advances provided by hydro-tRNAseq are applied towards the molecular diagnosis of a genetic neurodevelopmental syndrome, caused by mutations in the tRNA processing factor CLP1. Finally, I harness this novel experimental and computational expertise towards the identification of the endonuclease complex C3PO as a novel processing factor of human tRNAs. I carry out a transcriptome-wide analysis of C3PO targets, identify its binding sites and motifs, and provide insights into its biochemical and biological functions

Systems Biology Reveals MicroRNA-Mediated Gene Regulation

MicroRNAs (miRNAs) are members of the small non-coding RNAs, which are principally known for their functions as post-transcriptional regulators of target genes. Regulation by miRNAs is triggered by the translational repression or degradation of their complementary target messenger RNAs (mRNAs). The growing number of reported miRNAs and the estimate that hundreds or thousands of genes are regulated by them suggest a magnificent gene regulatory network in which these molecules are embedded. Indeed, recent reports have suggested critical roles for miRNAs in various biological functions, such as cell differentiation, development, oncogenesis, and the immune responses, which are mediated by systems-wide changes in gene expression profiles. Therefore, it is essential to analyze this complex regulatory network at the transcriptome and proteome levels, which should be possible with approaches that include both high-throughput experiments and computational methodologies. Here, we introduce several systems-level approaches that have been applied to miRNA research, and discuss their potential to reveal miRNA-guided gene regulatory systems and their impacts on biological functions