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PIWI-interacting RNAs, or piRNAs, are the most abundant short non-coding RNAs. More than 20,000 genes have been found in humans that code for piRNAs while only 2000 genes have been found for miRNAs. piRNAs can act at the transcriptional and post-transcriptional levels and have a vital role in silencing transposable elements present in germ cells. They are also involved in epigenetic silencing and activation. Previously, they were thought to function only in germ cells but new evidence suggests...
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Isolation of Cognate RNA-protein Complexes from Cells Using Oligonucleotide-directed Elution
10:53

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Published on: January 16, 2017

Dos complejos de RNAi, RITS y RDRC, interactúan físicamente y se localizan en ARN centroméricos no codificantes.

Mohammad R Motamedi1, André Verdel, Serafin U Colmenares

  • 1Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA.

Cell
|December 21, 2004
PubMed
Resumen

El complejo de ARN polimerasa dirigido por ARN (RDRC) se une física y funcionalmente con el complejo de silenciamiento transcripcional inducido por ARN (RITS). Esta interacción es crucial para el ensamblaje de la heterocromatina dependiente del siRNA en la levadura de fisión.

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Área de la Ciencia:

  • La epigenética es la epigenética.
  • La interferencia del ARN (RNAi) es una interferencia del ARN.
  • Biología molecular La biología molecular.

Sus antecedentes:

  • El ensamblaje de la heterocromatina mediada por ARNi requiere el complejo RITS y Rdp1.1.
  • La función precisa y las interacciones de Rdp1 en este proceso no se comprendieron completamente.

Objetivo del estudio:

  • Para dilucidar las interacciones moleculares y el significado funcional de Rdp1 en el ensamblaje de la heterocromatina.
  • Para caracterizar el complejo formado por Rdp1 y sus proteínas asociadas.

Principales métodos:

  • Co-inmunoprecipitación para identificar complejos de proteínas que interactúan.
  • Análisis de la localización de proteínas mediante microscopía.
  • El perfil del siRNA en las cepas knockout.
  • Pruebas para la actividad de la ARN polimerasa dirigida por ARN.

Principales resultados:

  • Rdp1 forma un complejo (RDRC) con Hrr1 (ARN helicasa) y Cid12 (poliA polimerasa) con actividad de ARN dirigida por ARN polimerasa.
  • RDRC interactúa con RITS, lo que requiere Dicer (Dcr1) y Clr4.
  • Tanto RDRC como RITS se localizan en el núcleo y se asocian con ARN centroméricos de una manera dependiente de Dcr1.
  • La pérdida de Rdp1, Hrr1 o Cid12 da como resultado que los RITS carezcan de siRNA y no logren localizarse a las repeticiones centroméricas.

Conclusiones:

  • Existe un vínculo físico y funcional entre Rdp1 y el complejo RITS.
  • Los ARN no codificantes sirven como plataforma para la localización dependiente de siRNA de la maquinaria de RNAi a regiones genómicas específicas.
  • Este estudio revela un nuevo mecanismo para la formación de heterocromatina mediada por la actividad de la ARN polimerasa dirigida por ARN y RITS.