RESUMO
Understanding genome organization and gene regulation requires insight into RNA transcription, processing and modification. We adapted nanopore direct RNA sequencing to examine RNA from a wild-type accession of the model plant Arabidopsis thaliana and a mutant defective in mRNA methylation (m6A). Here we show that m6A can be mapped in full-length mRNAs transcriptome-wide and reveal the combinatorial diversity of cap-associated transcription start sites, splicing events, poly(A) site choice and poly(A) tail length. Loss of m6A from 3' untranslated regions is associated with decreased relative transcript abundance and defective RNA 3' end formation. A functional consequence of disrupted m6A is a lengthening of the circadian period. We conclude that nanopore direct RNA sequencing can reveal the complexity of mRNA processing and modification in full-length single molecule reads. These findings can refine Arabidopsis genome annotation. Further, applying this approach to less well-studied species could transform our understanding of what their genomes encode.
Assuntos
Adenosina/análogos & derivados , Arabidopsis/genética , Processamento Pós-Transcricional do RNA , RNA Mensageiro/genética , RNA de Plantas/genética , Análise de Sequência de RNA , Adenosina/metabolismo , Arabidopsis/metabolismo , Perfilação da Expressão Gênica , Metilação , Nanoporos , Poli A/genética , Poli A/metabolismo , Capuzes de RNA , Splicing de RNA , RNA Mensageiro/química , RNA Mensageiro/metabolismo , RNA de Plantas/química , RNA de Plantas/metabolismo , RNA não Traduzido/química , RNA não Traduzido/genéticaRESUMO
Estimation of the period length of time-course data from cyclical biological processes, such as those driven by the circadian pacemaker, is crucial for inferring the properties of the biological clock found in many living organisms. We propose a methodology for period estimation based on spectrum resampling (SR) techniques. Simulation studies show that SR is superior and more robust to non-sinusoidal and noisy cycles than a currently used routine based on Fourier approximations. In addition, a simple fit to the oscillations using linear least squares is available, together with a non-parametric test for detecting changes in period length which allows for period estimates with different variances, as frequently encountered in practice. The proposed methods are motivated by and applied to various data examples from chronobiology.