RESUMO
In planarian flatworms, the piRNA pathway is operated by three PIWI proteins, termed SMEDWI-1, SMEDWI-2, and SMEDWI-3 (SMEDWI = Schmidtea mediterranea PIWI). The interplay between these three PIWI proteins and their associated small noncoding RNAs, termed piRNAs, fuels the outstanding regenerative abilities of planarians, enables tissue homeostasis, and, ultimately, ensures animal survival. As the molecular targets of PIWI proteins are determined by the sequences of their co-bound piRNAs, it is imperative to identify these sequences by next-generation sequencing applications. Following sequencing, the genomic targets and the regulatory potential of the isolated piRNA populations need to be uncovered. To that end, here we present a bioinformatics analysis pipeline for processing and systematic characterization of planarian piRNAs. The pipeline includes steps for the removal of PCR duplicates based on unique molecular identifier (UMI) sequences, and it accounts for piRNA multimapping to different loci in the genome. Importantly, our protocol also includes a fully automated pipeline that is freely available at GitHub. Together with the piRNA isolation and library preparation protocol (see accompanying chapter), the presented computational pipeline enables researchers to explore the functional role of the piRNA pathway in flatworm biology.
Assuntos
Biologia Computacional , Genoma , RNA de Interação com Piwi , Planárias , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , Biologia Computacional/métodos , Genoma/genética , Estudo de Associação Genômica Ampla , RNA de Interação com Piwi/genética , Planárias/genética , Internet , SoftwareRESUMO
Enhancer RNAs (eRNAs) are long non-coding RNAs that originate from enhancers. Although eRNA transcription is a canonical feature of activated enhancers, the molecular features required for eRNA function and the mechanism of how eRNAs impinge on target gene transcription have not been established. Thus, using eRNA-dependent RNA polymerase II (Pol II) pause release as a model, we here investigate the requirement of sequence, structure and length of eRNAs for their ability to stimulate Pol II pause release by detaching NELF from paused Pol II. We find eRNAs not to exert their function through common structural or sequence motifs. Instead, eRNAs that exhibit a length >200 nucleotides and that contain unpaired guanosines make multiple, allosteric contacts with NELF subunits -A and -E to trigger efficient NELF release. By revealing the molecular determinants of eRNA function, our study establishes eRNAs as an important player in Pol II pause release, and it provides new insight into the regulation of metazoan transcription.
Assuntos
RNA Polimerase II , RNA Longo não Codificante , Animais , Elementos Facilitadores Genéticos , Regulação da Expressão Gênica , Regiões Promotoras Genéticas , RNA Polimerase II/metabolismo , RNA Longo não Codificante/fisiologia , Sequências Reguladoras de Ácido Nucleico , Transcrição GênicaRESUMO
Lung cancer has currently the highest cancer-related mortality rate worldwide. MicroRNAs (miRNAs) are small noncoding RNAs that play a fundamental role in gene expression and are linked to disease progression of different cancer types such as lung cancer. However, functional characterization is made difficult by the fact that miRNAs generally regulate several mRNA interaction partners, resulting in complex regulatory networks. Thus, analysis of the network biology of miRNAs is essential for comprehensive understanding of their regulatory effects in lung cancer. A deeper understanding of miRNA networks in cancer could finally serve as a basis for the development of new therapeutic interventions. Here, we present a systems biology approach to analyze regulatory miRNA interaction networks to get better insight into their function.