LIM-domain proteins, LIMD1, Ajuba, and WTIP are required for microRNA-mediated gene silencing

Abstract

In recent years there have been major advances with respect to the identification of the protein components and mechanisms of micro-RNA (miRNA) mediated silencing. However, the complete and precise repertoire of components and mechanism(s) of action remain to be fully elucidated. Herein we reveal the identification of a family of three LIM domain-containing proteins, LIMD1, Ajuba and WTIP (Ajuba LIM proteins) as novel mammalian processing body (P-body) components, which highlight a novel mechanism of miRNA-mediated gene silencing. Furthermore, we reveal that LIMD1, Ajuba, and WTIP bind to Ago1∕2, RCK, Dcp2, and eIF4E in vivo, that they are required for miRNA-mediated, but not siRNA-mediated gene silencing and that all three proteins bind to the mRNA 5′ m 7 GTP cap-protein complex. Mechanistically, we propose the Ajuba LIM proteins interact with the m 7 GTP cap structure via a specific interaction with eIF4E that prevents 4EBP1 and eIF4G interaction. In addition, these LIM-domain proteins facilitate miRNA-mediated gene silencing by acting as an essential molecular link between the translationally inhibited eIF4E-m 7 GTP-5 0 cap and Ago1∕2 within the miRISC complex attached to the 3′-UTR of mRNA, creating an inhibitory closed-loop complex.argonaute | eIF4E | tumor suppressor | P-bodies | m 7 GTP cap M icroRNAs (miRNAs) are small noncoding RNAs that play important roles in a wide range of biological processes including development, cellular differentiation, proliferation, apoptosis, and cancer (1). To execute their regulatory functions miRNAs assemble together with the Argonaute (Ago) proteins into miRNA induced silencing complexes (miRISCs) (2). miRNAs within these complexes guide the bound Ago proteins to fully or partially complementary mRNA target sequences, resulting in mRNAs that are then silenced posttranscriptionally (2). Despite major advances in identifying key components of this pathway the complete protein repertoire and mechanism(s) of miRNA-mediated silencing remain to be fully elucidated. Here we show a distinct mechanism of miRNA-mediated silencing through the identification of three unique mammalian processing body (P-body) associated proteins, LIMD1, Ajuba, and WTIP (Ajuba LIM proteins) (3). The Ajuba LIM proteins are part of the larger Zyxin family, characterized by triple tandem arrayed c-terminal zinc-finger LIM domains. We demonstrate that these three LIM-domain containing proteins (4) interact with components of RNA induced silencing complexes (RISC) as well as eIF4E and the mRNA m 7 GTP cap-protein complex and are required for miRNA-mediated gene silencing. We propose a model whereby the LIMD1, Ajuba, and WTIP proteins enable miRNAmediated silencing by facilitating a simultaneous association between the translationally repressed m 7 GTP cap structure and an active miRISC complex attached to the 3′-UTR of mRNA. We propose this role in miRNA-mediated regulation represents a tumor suppressive function of Ajuba LIM proteins, specifically LIMD1, which has previously been validated as a bona fide ...

Literature review of baseline information to support the risk assessment of RNAi‐based GM plants

“…There are additional RISC interactions, which are important for miRNA-mediated silencing, such as DDX6/RCKp54 (Chu and Rana, 2006;Mathys et al, 2014;Kuzuoglu-Ozturk et al, 2016) or LIM domain proteins LIMD1, Ajuba, and WTIP, which are required for miRNA-mediated, but not siRNAmediated gene silencing (James et al, 2010). According to the model, the LIM proteins facilitate miRNA-mediated gene silencing by creating an inhibitory closed-loop complex where they bridge the translationally inhibited cap structure and AGO1/2 within the miRISC complex bound to the 3'-UTR (James et al, 2010).…”

“…P-bodies (reviewed in detail Jain and Parker, 2013) are distinct cytoplasmic foci, which contain proteins associated miRNA-mediated repression (Liu et al, 2005b;Rehwinkel et al, 2005;Yu et al, 2005;Behm-Ansmant et al, 2006;Chu and Rana, 2006;Zhou et al, 2009;James et al, 2010;Johnston et al, 2010;Pare et al, 2011;Chahar et al, 2013;Ozgur and Stoecklin, 2013). P-body association has been observed for the mature RISC components and RNA degradation pathway proteins but not for Dicer or TARBP2, indicating that P-bodies are associated with miRNA-mediated suppression but not biogenesis.…”

“…According to the model, the LIM proteins facilitate miRNA-mediated gene silencing by creating an inhibitory closed-loop complex where they bridge the translationally inhibited cap structure and AGO1/2 within the miRISC complex bound to the 3'-UTR (James et al, 2010). The list of proteins associated with RISC or the target repression is longer, and includes, for example, also Pumilio/FBF , a miRNA targeting cofactor (Friend et al, 2012) or eIF4GI (Ryu et al, 2013).…”

The LIM protein Ajuba promotes adipogenesis by enhancing PPARγ and p300/CBP interaction

“…[16][17][18]22 Ajuba functions as a scaffold involving assembly of multiple protein complexes to regulate cell adhesion, migration, mitosis, microRNA maturation, cell differentiation and tissue development. [16][17][18][19][20][21][22][23][24][25][26][27][28][29][30][31] Ajuba can directly participate in transcriptional regulation where it serves as a transcriptional co-repressor and downregulate gene expression. We lately identified Ajuba as a co-repressor for Snail and as an essential regulator of mesenchymal-epithelial transition and metastasis.…”

On the Connection between RNAi and Heterochromatin at Centromeres

“…Interestingly, a family of LIM-domain proteins was recently found to be required for miRNA-based silencing in human cells. These human LIM proteins interact with both Ago proteins and translation initiation complexes, thereby facilitating miRNA-mediated translational repression of target transcripts (James et al 2010). These findings suggest a conserved role for LIM-domain proteins in linking Ago-containing RNAi effector complexes to different gene regulation activities.…”