Showing papers by "Nikolaus Rajewsky published in 2014"

circBase: a database for circular RNAs

Abstract: Recently, several laboratories have reported thousands of circular RNAs (circRNAs) in animals. Numerous circRNAs are highly stable and have specific spatiotemporal expression patterns. Even though a function for circRNAs is unknown, these features make circRNAs an interesting class of RNAs as possible biomarkers and for further research. We developed a database and website, "circBase," where merged and unified data sets of circRNAs and the evidence supporting their expression can be accessed, downloaded, and browsed within the genomic context. circBase also provides scripts to identify known and novel circRNAs in sequencing data. The database is freely accessible through the web server at http://www.circbase.org/.

Identification of small ORFs in vertebrates using ribosome footprinting and evolutionary conservation

Abstract: Identification of the coding elements in the genome is a fundamental step to understanding the building blocks of living systems. Short peptides (< 100 aa) have emerged as important regulators of development and physiology, but their identification has been limited by their size. We have leveraged the periodicity of ribosome movement on the mRNA to define actively translated ORFs by ribosome footprinting. This approach identifies several hundred translated small ORFs in zebrafish and human. Computational prediction of small ORFs from codon conservation patterns corroborates and extends these findings and identifies conserved sequences in zebrafish and human, suggesting functional peptide products (micropeptides). These results identify micropeptide-encoding genes in vertebrates, providing an entry point to define their function in vivo.

Modules, networks and systems medicine for understanding disease and aiding diagnosis

Abstract: Many common diseases, such as asthma, diabetes or obesity, involve altered interactions between thousands of genes. High-throughput techniques (omics) allow identification of such genes and their products, but functional understanding is a formidable challenge. Network-based analyses of omics data have identified modules of disease-associated genes that have been used to obtain both a systems level and a molecular understanding of disease mechanisms. For example, in allergy a module was used to find a novel candidate gene that was validated by functional and clinical studies. Such analyses play important roles in systems medicine. This is an emerging discipline that aims to gain a translational understanding of the complex mechanisms underlying common diseases. In this review, we will explain and provide examples of how network-based analyses of omics data, in combination with functional and clinical studies, are aiding our understanding of disease, as well as helping to prioritize diagnostic markers or therapeutic candidate genes. Such analyses involve significant problems and limitations, which will be discussed. We also highlight the steps needed for clinical implementation.

RNA-binding protein RBM20 represses splicing to orchestrate cardiac pre-mRNA processing

Abstract: Mutations in the gene encoding the RNA-binding protein RBM20 have been implicated in dilated cardiomyopathy (DCM), a major cause of chronic heart failure, presumably through altering cardiac RNA splicing. Here, we combined transcriptome-wide crosslinking immunoprecipitation (CLIP-seq), RNA-seq, and quantitative proteomics in cell culture and rat and human hearts to examine how RBM20 regulates alternative splicing in the heart. Our analyses revealed the presence of a distinct RBM20 RNA-recognition element that is predominantly found within intronic binding sites and linked to repression of exon splicing with RBM20 binding near 3' and 5' splice sites. Proteomic analysis determined that RBM20 interacts with both U1 and U2 small nuclear ribonucleic particles (snRNPs) and suggested that RBM20-dependent splicing repression occurs through spliceosome stalling at complex A. Direct RBM20 targets included several genes previously shown to be involved in DCM as well as genes not typically associated with this disease. In failing human hearts, reduced expression of RBM20 affected alternative splicing of several direct targets, indicating that differences in RBM20 expression may affect cardiac function. Together, these findings identify RBM20-regulated targets and provide insight into the pathogenesis of human heart failure.

Global characterization of the oocyte-to-embryo transition in Caenorhabditis elegans uncovers a novel mRNA clearance mechanism

Abstract: The oocyte-to-embryo transition (OET) is thought to be mainly driven by post-transcriptional gene regulation. However, expression of both RNAs and proteins during the OET has not been comprehensively assayed. Furthermore, specific molecular mechanisms that regulate gene expression during OET are largely unknown. Here, we quantify and analyze transcriptome-wide, expression of mRNAs and thousands of proteins in Caenorhabditis elegans oocytes, 1-cell, and 2-cell embryos. This represents a first comprehensive gene expression atlas during the OET in animals. We discovered a first wave of degradation in which thousands of mRNAs are cleared shortly after fertilization. Sequence analysis revealed a statistically highly significant presence of a polyC motif in the 3' untranslated regions of most of these degraded mRNAs. Transgenic reporter assays demonstrated that this polyC motif is required and sufficient for mRNA degradation after fertilization. We show that orthologs of human polyC-binding protein specifically bind this motif. Our data suggest a mechanism in which the polyC motif and binding partners direct degradation of maternal mRNAs. Our data also indicate that endogenous siRNAs but not miRNAs promote mRNA clearance during the OET.

Paternal RNA contributions in the Caenorhabditis elegans zygote

Abstract: Development of the early embryo is thought to be mainly driven by maternal gene products and post-transcriptional gene regulation. Here, we used metabolic labeling to show that RNA can be transferred by sperm into the oocyte upon fertilization. To identify genes with paternal expression in the embryo, we performed crosses of males and females from divergent Caenorhabditis elegans strains. RNA sequencing of mRNAs and small RNAs in the 1-cell hybrid embryo revealed that about one hundred sixty paternal mRNAs are reproducibly expressed in the embryo and that about half of all assayed endogenous siRNAs and piRNAs are also of paternal origin. Together, our results suggest an unexplored paternal contribution to early development.

Binding site discovery from nucleic acid sequences by discriminative learning of hidden Markov models

Abstract: We present a discriminative learning method for pattern discovery of binding sites in nucleic acid sequences based on hidden Markov models. Sets of positive and negative example sequences are mined for sequence motifs whose occurrence frequency varies between the sets. The method offers several objective functions, but we concentrate on mutual information of condition and motif occurrence. We perform a systematic comparison of our method and numerous published motif-finding tools. Our method achieves the highest motif discovery performance, while being faster than most published methods. We present case studies of data from various technologies, including ChIP-Seq, RIP-Chip and PAR-CLIP, of embryonic stem cell transcription factors and of RNA-binding proteins, demonstrating practicality and utility of the method. For the alternative splicing factor RBM10, our analysis finds motifs known to be splicing-relevant. The motif discovery method is implemented in the free software package Discrover. It is applicable to genome- and transcriptome-scale data, makes use of available repeat experiments and aside from binary contrasts also more complex data configurations can be utilized.

Mixed messages: Re-initiation factors regulate translation of animal mRNAs.

Abstract: When ribosomes encounter upstream open reading frames (uORFs) during scanning of the 5′ untranslated region (5′ UTR), translation of the downstream ORF requires re-initiation. In a recent paper in Nature, Schleich et al. describe metazoan factors which specifically promote re-initiation.