RESUMEN
Thousands of long intergenic non-coding RNAs (lincRNAs) are transcribed throughout the vertebrate genome. A subset of lincRNAs enriched in developing brains have recently been found to contain cryptic open-reading frames and are speculated to encode micropeptides. However, systematic identification and functional assessment of these transcripts have been hindered by technical challenges caused by their small size. Here, we show that two putative lincRNAs (linc-mipep, also called lnc-rps25, and linc-wrb) encode micropeptides with homology to the vertebrate-specific chromatin architectural protein, Hmgn1, and demonstrate that they are required for development of vertebrate-specific brain cell types. Specifically, we show that NMDA receptor-mediated pathways are dysregulated in zebrafish lacking these micropeptides and that their loss preferentially alters the gene regulatory networks that establish cerebellar cells and oligodendrocytes - evolutionarily newer cell types that develop postnatally in humans. These findings reveal a key missing link in the evolution of vertebrate brain cell development and illustrate a genetic basis for how some neural cell types are more susceptible to chromatin disruptions, with implications for neurodevelopmental disorders and disease.
Asunto(s)
ARN Largo no Codificante , Animales , Humanos , ARN Largo no Codificante/genética , Cromatina , Pez Cebra/genética , Pez Cebra/metabolismo , Diferenciación Celular/genética , MicropéptidosRESUMEN
Protein coding genes were originally identified with sequence-based definitions that included a 100-codon cutoff to avoid annotating irrelevant open reading frames. However, many active proteins contain less than 100 amino acids. Indeed, functional genetics, ribosome profiling, and proteomic profiling have identified many short, translated open reading frames, including those with biologically active peptide products (microproteins). Yet, functions for most of these peptide products remain unknown. Because microproteins often act as key signals or fine-tune processes, animal development has already revealed functions for a handful of microproteins and provides an ideal context to uncover additional microprotein functions. However, many mRNAs during early development are maternally provided and hinder targeted mutagenesis approaches to characterize developmental microprotein functions. The recently established, RNA-targeting CRISPR-Cas13d system in zebrafish overcomes this barrier and produces potent knockdown of targeted mRNA, including maternally provided mRNA, and enables flexible, efficient interrogation of microprotein functions in animal development.
RESUMEN
Messenger RNA (mRNA) stability substantially impacts steady-state gene expression levels in a cell. mRNA stability is strongly affected by codon composition in a translation-dependent manner across species, through a mechanism termed codon optimality. We have developed iCodon ( www.iCodon.org ), an algorithm for customizing mRNA expression through the introduction of synonymous codon substitutions into the coding sequence. iCodon is optimized for four vertebrate transcriptomes: mouse, human, frog, and fish. Users can predict the mRNA stability of any coding sequence based on its codon composition and subsequently generate more stable (optimized) or unstable (deoptimized) variants encoding for the same protein. Further, we show that codon optimality predictions correlate with both mRNA stability using a massive reporter library and expression levels using fluorescent reporters and analysis of endogenous gene expression in zebrafish embryos and/or human cells. Therefore, iCodon will benefit basic biological research, as well as a wide range of applications for biotechnology and biomedicine.
Asunto(s)
Biosíntesis de Proteínas , Pez Cebra , Animales , Codón/genética , Humanos , Ratones , Proteínas/metabolismo , Estabilidad del ARN/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Pez Cebra/genética , Pez Cebra/metabolismoRESUMEN
BACKGROUND: The regulation of messenger RNA (mRNA) stability has a profound impact on gene expression dynamics during embryogenesis. For example, in animals, maternally deposited mRNAs are degraded after fertilization to enable new developmental trajectories. Regulatory sequences in 3' untranslated regions (3'UTRs) have long been considered the central determinants of mRNA stability. However, recent work indicates that the coding sequence also possesses regulatory information. Specifically, translation in cis impacts mRNA stability in a codon-dependent manner. However, the strength of this mechanism during embryogenesis, as well as its relationship with other known regulatory elements, such as microRNA, remains unclear. RESULTS: Here, we show that codon composition is a major predictor of mRNA stability in the early embryo. We show that this mechanism works in combination with other cis-regulatory elements to dictate mRNA stability in zebrafish and Xenopus embryos as well as in mouse and human cells. Furthermore, we show that microRNA targeting efficacy can be affected by substantial enrichment of optimal (stabilizing) or non-optimal (destabilizing) codons. Lastly, we find that one microRNA, miR-430, antagonizes the stabilizing effect of optimal codons during early embryogenesis in zebrafish. CONCLUSIONS: By integrating the contributions of different regulatory mechanisms, our work provides a framework for understanding how combinatorial control of mRNA stability shapes the gene expression landscape.
Asunto(s)
Codón , Estabilidad del ARN , ARN Mensajero/genética , Secuencias Reguladoras de Ácidos Nucleicos , Regiones no Traducidas 3' , Animales , Desarrollo Embrionario/genética , Regulación del Desarrollo de la Expresión Génica , Humanos , Ratones , MicroARNs/metabolismo , Modelos Genéticos , Xenopus/genética , Pez Cebra/genéticaRESUMEN
mRNA translation decodes nucleotide into amino acid sequences. However, translation has also been shown to affect mRNA stability depending on codon composition in model organisms, although universality of this mechanism remains unclear. Here, using three independent approaches to measure exogenous and endogenous mRNA decay, we define which codons are associated with stable or unstable mRNAs in human cells. We demonstrate that the regulatory information affecting mRNA stability is encoded in codons and not in nucleotides. Stabilizing codons tend to be associated with higher tRNA levels and higher charged/total tRNA ratios. While mRNAs enriched in destabilizing codons tend to possess shorter poly(A)-tails, the poly(A)-tail is not required for the codon-mediated mRNA stability. This mechanism depends on translation; however, the number of ribosome loads into a mRNA modulates the codon-mediated effects on gene expression. This work provides definitive evidence that translation strongly affects mRNA stability in a codon-dependent manner in human cells.
Asunto(s)
Codón , Biosíntesis de Proteínas , Estabilidad del ARN , Línea Celular , HumanosRESUMEN
Expression of tobacco mosaic virus (TMV) coat protein (CP) restricts virus disassembly and alters the accumulation of the movement protein (MP). To characterize the role of structure of transgenic CP in regulating virus disassembly and production of MP, we generated CPs with mutations at residues Glu50 and Asp77, located in the interface between juxtaposed CP subunits. In transgenic Nicotiana tabacum and BY-2 cells, three categories of coat protein-mediated resistance (CP-MR) levels were identified: wild-type CP-MR; elevated CP-MR; and no CP-MR. Mutant CPs that interfered with the accumulation of virus replication complexes conferred very high levels of protection to TMV, except by CP(E50D) which provided no protection in the systemic host (Xanthi-nn) but high CP-MR in the local lesion host (Xanthi-NN). In transgenic BY-2 cells CP(E50D) strongly reduced accumulation of MP:GFP. In general, there was a strong correlation between the capacity for CP to assemble to pseudovirions and CP-MR, while there was not strong correlation with packaging viral RNA and CP-MR. The data demonstrate that interference with one or more steps in virus infection and replication by wild type and mutant CP determine the degree of CP-MR.