RESUMEN
Deep sequencing now provides detailed snapshots of ribosome occupancy on mRNAs. We leverage these data to parameterize a computational model of translation, keeping track of every ribosome, tRNA, and mRNA molecule in a yeast cell. We determine the parameter regimes in which fast initiation or high codon bias in a transgene increases protein yield and infer the initiation rates of endogenous Saccharomyces cerevisiae genes, which vary by several orders of magnitude and correlate with 5' mRNA folding energies. Our model recapitulates the previously reported 5'-to-3' ramp of decreasing ribosome densities, although our analysis shows that this ramp is caused by rapid initiation of short genes rather than slow codons at the start of transcripts. We conclude that protein production in healthy yeast cells is typically limited by the availability of free ribosomes, whereas protein production under periods of stress can sometimes be rescued by reducing initiation or elongation rates.
Asunto(s)
Modelos Genéticos , Biosíntesis de Proteínas , Saccharomyces cerevisiae/genética , Codón/genética , Cadenas de Markov , ARN Mensajero/metabolismo , ARN de Transferencia/metabolismo , Ribosomas/metabolismoRESUMEN
Imprinted gene clusters are regulated by long noncoding RNAs (lncRNAs), CCCTC binding factor (CTCF)-mediated boundaries, and DNA methylation. DIRAS3 (also known as ARH1 or NOEY1) is an imprinted gene encoding a protein belonging to the RAS superfamily of GTPases and is located within an intron of a lncRNA called GNG12-AS1. In this study, we investigated whether GNG12-AS1 is imprinted and coregulated with DIRAS3. We report that GNG12-AS1 is coexpressed with DIRAS3 in several tissues and coordinately downregulated with DIRAS3 in breast cancers. GNG12-AS1 has several splice variants, all of which initiate from a single transcription start site. In placenta tissue and normal cell lines, GNG12-AS1 is biallelically expressed but some isoforms are allele-specifically spliced. Cohesin plays a role in allele-specific splicing of GNG12-AS1. In breast cancer cell lines with loss of DIRAS3 imprinting, DIRAS3 and GNG12-AS1 are silenced in cis and the remaining GNG12-AS1 transcripts are predominantly monoallelic. The GNG12-AS1 locus, which includes DIRAS3, provides an example of imprinted cotranscriptional splicing and a potential model system for studying the long-range effects of CTCF-cohesin binding on splicing and transcriptional interference.
Asunto(s)
Neoplasias de la Mama/genética , Proteínas de Ciclo Celular/genética , Cromatina/metabolismo , Proteínas Cromosómicas no Histona/genética , Impresión Genómica , ARN Largo no Codificante/genética , Proteínas de Unión al GTP rho/genética , Alelos , Empalme Alternativo , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Proteínas Cromosómicas no Histona/metabolismo , Metilación de ADN , Femenino , Regulación de la Expresión Génica , Humanos , Intrones , Placenta/citología , Placenta/metabolismo , Embarazo , ARN Largo no Codificante/metabolismo , Sitio de Iniciación de la Transcripción , Transcripción Genética , Proteínas de Unión al GTP rho/metabolismo , CohesinasRESUMEN
Long noncoding RNAs (lncRNAs) regulate gene expression via their RNA product or through transcriptional interference, yet a strategy to differentiate these two processes is lacking. To address this, we used multiple small interfering RNAs (siRNAs) to silence GNG12-AS1, a nuclear lncRNA transcribed in an antisense orientation to the tumour-suppressor DIRAS3. Here we show that while most siRNAs silence GNG12-AS1 post-transcriptionally, siRNA complementary to exon 1 of GNG12-AS1 suppresses its transcription by recruiting Argonaute 2 and inhibiting RNA polymerase II binding. Transcriptional, but not post-transcriptional, silencing of GNG12-AS1 causes concomitant upregulation of DIRAS3, indicating a function in transcriptional interference. This change in DIRAS3 expression is sufficient to impair cell cycle progression. In addition, the reduction in GNG12-AS1 transcripts alters MET signalling and cell migration, but these are independent of DIRAS3. Thus, differential siRNA targeting of a lncRNA allows dissection of the functions related to the process and products of its transcription.