The majority of transcripts in the squid nervous system are extensively recoded by A-to-I RNA editing
Shahar Alon, Sandra C Garrett, Erez Y Levanon, Sara Olson, Brenton R Graveley, Joshua J C Rosenthal, Eli Eisenberg Corresponding Author
Tel Aviv University, Israel; University of Connecticut Health Center, United States; Bar-Ilan University, Israel; University of Puerto Rico Medical Sciences Campus, Puerto Rico
DOI: http://dx.doi.org/10.7554/eLife.05198
Published January 8, 2015
Abstract
RNA editing by adenosine deamination alters genetic information from the genomic blueprint. When it recodes mRNAs, it gives organisms the option to express diverse, functionally distinct, protein isoforms. All eumetazoans, from cnidarians to humans, express RNA editing enzymes. However, transcriptome-wide screens have only uncovered about 25 transcripts harboring conserved recoding RNA editing sites in mammals and several hundred recoding sites in Drosophila. These studies on few established models have led to the general assumption that recoding by RNA editing is extremely rare. Here we employ a novel bioinformatic approach with extensive validation to show that the squid Doryteuthis pealeii recodes proteins by RNA editing to an unprecedented extent. We identify 57,108 recoding sites in the nervous system, affecting the majority of the proteins studied. Recoding is tissue-dependent, and enriched in genes with neuronal and cytoskeletal functions, suggesting it plays an important role in brain physiology.
DOI: http://dx.doi.org/10.7554/eLife.05198.001
Excerpt/Excerto:
Introduction
The central dogma of biology maintains that genetic information passes faithfully from DNA to RNA to proteins; however, with the help of tools such as alternative splicing, organisms use RNA as a canvas to modify and enrich this flow of information. RNA editing by deamination of adenosine to inosine (A-to-I) is another process used to alter genetic information (Nishikura, 2010). Unlike alternative splicing, which shuffles relatively large regions of RNA, editing targets single bases in order to fine-tune protein function. Because inosine is interpreted as guanosine by the cellular machinery, this process can recode codons (Basilio et al., 1962). A-to-I RNA editing is catalyzed by the ADAR (adenosine deaminase that acts on RNA) family of enzymes. All eumetazoans, from cnidarians to mammals, express ADARs but the extent to which they use them to recode has been explored in few representatives (Nishikura, 2010).
Recent advances in DNA sequencing and the supporting computational analyses have permitted transcriptome-wide screens for RNA editing events. So far, such studies have been limited to organisms with a sequenced genome (Ramaswami et al., 2012, 2013). In general, these screens have looked for variation in RNA at positions that are invariant in the genome. In humans, inosine is abundant in RNA (Paul and Bass, 1998; Bazak et al., 2014), but almost all of it lies within transcribed repetitive elements in untranslated regions or introns (Nishikura, 2010). A compilation of recoding sites in human transcriptomes revealed 1183 events (Xu and Zhang, 2014), but most were observed in only a single sample. Individual searches (Danecek et al., 2012; Ramaswami et al., 2013) uncovered only 115 (non-repetitive) recoding events, and 53 in mice; 34 recoding sites are conserved across mammals (Pinto et al., 2014). In Drosophila, an order of magnitude more recoding sites have been identified, residing in about 3% of all messages (St Laurent et al., 2013). Although individual editing sites are clearly essential (Brusa et al., 1995), these data suggest that RNA editing is not a broadly used mechanism for proteome diversification.
However, anecdotal data suggest this assumption might not apply across the animal kingdom. For example, using traditional cloning methods, scores of recoding sites have been uncovered in a small number of squid and octopus transcripts encoding potassium channels, ADARs, and ion pumps (Garrett and Rosenthal, 2012a). As for most organisms, there are no genomes available for cephalopods. Here we apply a novel approach for editing site detection in the absence of a sequenced genome. We use it to comprehensively identify editing sites in the squid giant axon system and other areas of the nervous system. Surprisingly, almost 60% of all mRNAs studied harbor recoding events, and most at multiple sites. These data show orders of magnitude more recoding in the squid proteome than in any other species studied to date. In squid, editing is so pervasive that the central dogma should be modified to include this process. Our results open the possibility that extensive recoding is common in many organisms, rivaling alternative splicing as a means of creating functional diversity.
Results and discussion
To detect RNA editing sites in the squid nervous system, we generated millions of RNA and genomic DNA reads from an individual squid. Our method differed from previous approaches by using a de novo transcriptome as the point of reference instead of a genome (Figure 1A). The transcriptome was assembled from RNA-seq reads, and each nucleotide within it represents the consensus of many reads. If the majority of RNA reads were edited (‘strong’ editing sites), the transcriptome would differ from the genomic DNA and read ‘G’ where gDNA reads would show ‘A’ (the sequencing process identifies inosines as guanosines). We detected such sites by aligning DNA-seq reads to the transcriptome (Figure 1B). At positions where editing occurred in the minority of RNA-seq reads (‘weak’ editing sites), however, the transcriptome and the genomic DNA would be identical. These sites were detected by identifying variability in RNA-seq, but not DNA-seq, reads (Figure 1B). This general approach is applicable to all organisms that lack a sequenced genome.
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Dogma central em biologia evolucionária: DNA >>> RNA >>> Proteínas + mutações serviriam de material para a seleção natural "criar" novas espécies. E agora Darwin? O dogma central em biologia evolucionária foi para a lata do lixo da história da ciência. E o que restou, guru de Down? Ficou simplesmente o dogma que ainda vai continuar sendo "rezado" em muitos livros didáticos de biologia do ensino médio recomendados pelo MEC/SEMTEC/PNLEM, e em muitas universidades onde dizer que Darwin está nu é considerado pecado mortal acadêmico.
Darwin morreu! Viva Darwin!!!
