RESUMEN
MicroRNAs (miRNAs) are an abundant class of small regulatory RNAs that regulate the stability and translation of cognate mRNAs. Although an increasing number of porcine miRNAs has recently been identified, the full repertoire of miRNAs in pig remains to be elucidated. To identify porcine miRNAs potentially involved in myogenesis and adipogenesis, we constructed small RNA cDNA libraries from skeletal muscle and adipose tissue and identified 89 distinct miRNAs that are conserved in pig, of which 15 were new. Expression analysis of all newly identified and selected known porcine miRNAs revealed that some miRNAs were enriched in a tissue-specific manner, whereas others were expressed ubiquitously in the porcine tissues examined. Our results expand the number of known porcine miRNAs and provide useful information for further investigating the biological functions of miRNAs associated with growth and development of skeletal muscle or adipose tissue in pig.
Asunto(s)
Tejido Adiposo/metabolismo , MicroARNs/genética , Músculo Esquelético/metabolismo , Sus scrofa/genética , Animales , Secuencia de Bases , Clonación Molecular , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Genoma/genética , MicroARNs/química , MicroARNs/metabolismo , Datos de Secuencia Molecular , Familia de Multigenes/genética , Conformación de Ácido Nucleico , Precursores del ARN/química , Precursores del ARN/genética , Análisis de Secuencia de ARNRESUMEN
Small interfering RNAs (siRNAs) which mediate sequence-specific gene silencing through RNA interference have been harnessed for therapeutic applications. Here, we found combinations of modest 2'-ribose siRNA modifications that permit effective gene silencing and improve siRNA stability in human serum. Introduction of as few as two different or identical 2'-ribose modifications at a specific position of either strand, which has been previously suggested to suppress unintended off-target effects, enhances the thermal and thermodynamic stability of siRNA duplexes, and does not alter the overall A-form helical geometry that is required for silencing activity. Our results provide a useful template for the design for therapeutic siRNAs.
Asunto(s)
Interferencia de ARN , ARN Interferente Pequeño/sangre , ARN Interferente Pequeño/química , Western Blotting , Dicroismo Circular , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HeLa , Humanos , Desnaturalización de Ácido Nucleico , ARN Interferente Pequeño/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Ribosa/química , Suero/química , Termodinámica , TransfecciónRESUMEN
Small RNAs direct RNA-induced silencing complexes (RISCs) to regulate stability and translation of mRNAs. RISCs associated with target mRNAs often accumulate in discrete cytoplasmic foci known as GW-bodies. However, RISC proteins can associate with membrane compartments such as the Golgi and endoplasmic reticulum. Here, we show that GW-bodies are associated with late endosomes (multivesicular bodies, MVBs). Blocking the maturation of MVBs into lysosomes by loss of the tethering factor HPS4 (ref. 5) enhances short interfering RNA (siRNA)- and micro RNA (miRNA)-mediated silencing in Drosophila melanogaster and humans. It also triggers over-accumulation of GW-bodies. Blocking MVB formation by ESCRT (endosomal sorting complex required for transport) depletion results in impaired miRNA silencing and loss of GW-bodies. These results indicate that active RISCs are physically and functionally coupled to MVBs. We further show that MVBs promote the competence of RISCs in loading small RNAs. We suggest that the recycling of RISCs is promoted by MVBs, resulting in RISCs more effectively engaging with small RNA effectors and possibly target RNAs. It may provide a means to enhance the dynamics of RNA silencing in the cytoplasm.
Asunto(s)
Drosophila melanogaster/metabolismo , Endosomas/metabolismo , Silenciador del Gen , ARN Interferente Pequeño/metabolismo , Animales , Transporte Biológico , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citología , Células HeLa , Humanos , MicroARNs/metabolismo , Complejo Silenciador Inducido por ARN/metabolismo , UbiquitinaciónRESUMEN
MicroRNAs (miRNAs) are small regulatory RNAs that direct the posttranscriptional repression of cognate messenger RNAs. Despite increasing evidence for diverse roles of miRNAs in biological processes, little is known about miRNAs in pig. We describe the first experimental identification of porcine miRNAs by sequence analysis of a cDNA library of small RNAs from porcine fibroblast cells. We identified 25 distinct porcine miRNAs, of which 19 are previously unreported, and define 14 new miRNA families in pig. Most of the cloned miRNAs are expressed ubiquitously in all porcine tissues examined, whereas some miRNAs are expressed preferentially in specific tissues. Our results enrich the porcine miRNA database and provide useful information for investigating biological functions of miRNAs in pig.