RESUMEN
Seed dormancy is one of the most crucial process transitions in a plant's life cycle. Its timing is tightly controlled by the expression level of the Delay of Germination 1 gene (DOG1). DOG1 is the major quantitative trait locus for seed dormancy in Arabidopsis and has been shown to control dormancy in many other plant species. This is reflected by the evolutionary conservation of the functional short alternatively polyadenylated form of the DOG1 mRNA. Notably, the 3' region of DOG1, including the last exon that is not included in this transcript isoform, shows a high level of conservation at the DNA level, but the encoded polypeptide is poorly conserved. Here, we demonstrate that this region of DOG1 contains a promoter for the transcription of a noncoding antisense RNA, asDOG1, that is 5' capped, polyadenylated, and relatively stable. This promoter is autonomous and asDOG1 has an expression profile that is different from known DOG1 transcripts. Using several approaches we show that asDOG1 strongly suppresses DOG1 expression during seed maturation in cis, but is unable to do so in trans Therefore, the negative regulation of seed dormancy by asDOG1 in cis results in allele-specific suppression of DOG1 expression and promotes germination. Given the evolutionary conservation of the asDOG1 promoter, we propose that this cis-constrained noncoding RNA-mediated mechanism limiting the duration of seed dormancy functions across the Brassicaceae.
Asunto(s)
Proteínas de Arabidopsis/fisiología , Arabidopsis/fisiología , Regulación de la Expresión Génica de las Plantas , ARN sin Sentido/fisiología , Semillas/fisiología , Secuencia de Bases , Secuencia Conservada , Exones , Latencia en las Plantas , Regiones Promotoras Genéticas , ARN de Planta/fisiología , ARN no Traducido/fisiología , Transcripción GenéticaRESUMEN
DOG1 (Delay of Germination 1) is a key regulator of seed dormancy in Arabidopsis (Arabidopsis thaliana) and other plants. Interestingly, the C terminus of DOG1 is either absent or not conserved in many plant species. Here, we show that in Arabidopsis, DOG1 transcript is subject to alternative polyadenylation. In line with this, mutants in RNA 3' processing complex display weakened seed dormancy in parallel with defects in DOG1 proximal polyadenylation site selection, suggesting that the short DOG1 transcript is functional. This is corroborated by the finding that the proximally polyadenylated short DOG1 mRNA is translated in vivo and complements the dog1 mutant. In summary, our findings indicate that the short DOG1 protein isoform produced from the proximally polyadenylated DOG1 mRNA is a key player in the establishment of seed dormancy in Arabidopsis and characterizes a set of mutants in RNA 3' processing complex required for production of proximally polyadenylated functional DOG1 transcript.
Asunto(s)
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Arabidopsis/fisiología , Latencia en las Plantas/genética , Poliadenilación/genética , Semillas/fisiología , Secuencia de Aminoácidos , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Secuencia de Bases , Secuencia Conservada , Regulación de la Expresión Génica de las Plantas , Germinación , Datos de Secuencia Molecular , Mutación/genética , Fenotipo , Biosíntesis de Proteínas , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Procesamiento Postranscripcional del ARN , ARN Mensajero/genética , ARN Mensajero/metabolismo , Semillas/genéticaRESUMEN
Accurate tRNA processing is crucial for human mitochondrial genome expression, but the mechanisms of mt-tRNA cleavage and the key enzymes involved in this process are poorly characterized. At least two activities are required for proper mt-tRNA maturation: RNase P cleaving precursor molecules at the 5' end and tRNase Z at the 3' end. In human mitochondria only RNase P has been identified so far. Using RT-PCR and northern blot analyses we found that silencing of the human ELAC2 gene results in impaired 3' end of mt-tRNAs. We demonstrate this for several mitochondrial tRNAs, encoded on both mtDNA strands, including tRNA (Val) , tRNA (Lys) , tRNA (Arg) , tRNA (Gly) , tRNA (Leu(UUR)) and tRNA (Glu) . The silencing of the MRPP1 gene that encodes a subunit of mtRNase P resulted in inhibition of both 5' and 3' processing. We also demonstrate the double mitochondrial/nuclear localization of the ELAC2 protein using immunofluorescence. Our results indicate that ELAC2 functions as a tRNase Z in human mitochondria and suggest that mt-tRNase Z preferentially cleaves molecules already processed by the proteinaceous mtRNase P.
Asunto(s)
Mitocondrias/genética , Proteínas de Neoplasias/metabolismo , Procesamiento de Término de ARN 3'/genética , ARN de Transferencia/genética , ARN/metabolismo , Línea Celular Tumoral , Núcleo Celular/enzimología , Núcleo Celular/genética , Núcleo Celular/metabolismo , Endorribonucleasas/metabolismo , Células HEK293 , Células HeLa , Humanos , Mitocondrias/enzimología , Mitocondrias/metabolismo , Proteínas de Neoplasias/genética , ARN/genética , Interferencia de ARN , ARN Mitocondrial , ARN Interferente Pequeño , ARN de Transferencia/metabolismo , Ribonucleasa P/metabolismoRESUMEN
Protein complexes responsible for RNA degradation play important role in three key aspects of RNA metabolism: they control stability of physiologically functional transcripts, remove the unnecessary RNA processing intermediates and destroy aberrantly formed RNAs. In mitochondria the post-transcriptional events seem to play a major role in regulation of gene expression, therefore RNA turnover is of particular importance. Despite many years of research, the details of this process are still a challenge. This review summarizes emerging landscape of interplay between the Suv3p helicase (SUPV3L1, Suv3), poly(A) polymerase and polynucleotide phosphorylase in controlling RNA degradation in human mitochondria.
Asunto(s)
Mitocondrias/metabolismo , ARN/metabolismo , ARN Helicasas DEAD-box/metabolismo , Humanos , Técnicas In Vitro , Mitocondrias/genética , Modelos Biológicos , Poli U/metabolismo , Poliadenilación , Polinucleotido Adenililtransferasa/metabolismo , Polirribonucleótido Nucleotidiltransferasa/metabolismo , ARN/genética , Procesamiento Postranscripcional del ARN , Estabilidad del ARN , ARN de Hongos/genética , ARN de Hongos/metabolismo , ARN Mitocondrial , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMEN
The mechanism of human mitochondrial RNA turnover and surveillance is still a matter of debate. We have obtained a cellular model for studying the role of hSuv3p helicase in human mitochondria. Expression of a dominant-negative mutant of the hSUV3 gene which encodes a protein with no ATPase or helicase activity results in perturbations of mtRNA metabolism and enables to study the processing and degradation intermediates which otherwise are difficult to detect because of their short half-lives. The hSuv3p activity was found to be necessary in the regulation of stability of mature, properly formed mRNAs and for removal of the noncoding processing intermediates transcribed from both H and L-strands, including mirror RNAs which represent antisense RNAs transcribed from the opposite DNA strand. Lack of hSuv3p function also resulted in accumulation of aberrant RNA species, molecules with extended poly(A) tails and degradation intermediates truncated predominantly at their 3'-ends. Moreover, we present data indicating that hSuv3p co-purifies with PNPase; this may suggest participation of both proteins in mtRNA metabolism.