Importin 8 Is a Gene Silencing Factor that Targets Argonaute Proteins to Distinct mRNAs

Abstract

Small regulatory RNAs including small interfering RNAs (siRNAs) and microRNAs (miRNAs) guide Argonaute (Ago) proteins to specific target RNAs leading to mRNA destabilization or translational repression. Here, we report the identification of Importin 8 (Imp8) as a component of miRNA-guided regulatory pathways. We show that Imp8 interacts with Ago proteins and localizes to cytoplasmic processing bodies (P bodies), structures involved in RNA metabolism. Furthermore, we detect Ago2 in the nucleus of HeLa cells, and knockdown of Imp8 reduces the nuclear Ago2 pool. Using immunoprecipitations of Ago2-associated mRNAs followed by microarray analysis, we further demonstrate that Imp8 is required for the recruitment of Ago protein complexes to a large set of Ago2-associated target mRNAs, allowing for efficient and specific gene silencing. Therefore, we provide evidence that Imp8 is required for cytoplasmic miRNA-guided gene silencing and affects nuclear localization of Ago proteins.

Importin 8 Regulates the Transport of Mature MicroRNAs into the Cell Nucleus

“…2C), implying that the import of Ago2 into the nucleus is IPO8-dependent. Reduction of the nuclear Ago2 pool by IPO8 knockdown has also been demonstrated recently by Weinmann et al (16). The involvement of the Ago2 complex in IPO8-mediated cytoplasm-to-nucleus transport of miRNAs is further supported by the observation that TPF, a small molecule that specifically disrupts the association of Ago2 with miRNAs (14,17), blocked the cytoplasm-to-nuclear transport of all six nuclear miRNAs.…”

Systematic identification of long noncoding RNAs expressed during zebrafish embryogenesis

“…5). Consistent with this observation, MALAT1 has previously been shown to be associated with Ago2 (also known as EIF2C2), a known component of the sRNA processing machinery (Weinmann et al 2009). This analysis indicates that the large majority of our lncRNAs are not processed into sRNAs.…”

Nuclear microRNAs and their unconventional role in regulating non-coding RNAs

“…Robb et al fi rst showed the presence of active Argonaute proteins in nuclear compartments (Robb et al, 2005). In addition, Weinmann et al detected AGO2 in the nucleus of HeLa cells (Weinmann et al, 2009). Notably, it has been shown that Argonaute proteins are directly associated with nuclear miRNAs in Caenorhabditis elegans (C. elegans) (Zisoulis et al, 2012) and human cells (Hansen et al, 2011;Zisoulis et al, 2012).…”

“…These two papers suggest that Exportin-1 may serve as a pore for the translocation of both miRNAs and Argonaute proteins from the cytoplasm to the nucleus. In another interesting study, Weinmann et al showed that Importin-8 interacted with human Argonaute proteins in a Ran-dependent manner (Weinmann et al, 2009). Additionally, they found that upon Importin-8 knockdown, the localization of AGO2 was shifted from the nucleus to the cytoplasm, whereas the total amount of AGO2 remained unaffected (Weinmann et al, 2009).…”

“…In another interesting study, Weinmann et al showed that Importin-8 interacted with human Argonaute proteins in a Ran-dependent manner (Weinmann et al, 2009). Additionally, they found that upon Importin-8 knockdown, the localization of AGO2 was shifted from the nucleus to the cytoplasm, whereas the total amount of AGO2 remained unaffected (Weinmann et al, 2009). The results suggest that Importin-8 can infl uence nuclear import of AGO2 in human cells.…”