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 ...

There are three prolyl hydroxylases (PHD1, 2 and 3) that regulate the hypoxia-inducible factors (HIFs), the master transcriptional regulators that respond to changes in intracellular O(2) tension. In high O(2) tension (normoxia) the PHDs hydroxylate two conserved proline residues on HIF-1α, which leads to binding of the von Hippel-Lindau (VHL) tumour suppressor, the recognition component of a ubiquitin-ligase complex, initiating HIF-1α ubiquitylation and degradation. However, it is not known whether PHDs and VHL act separately to exert their enzymatic activities on HIF-1α or as a multiprotein complex. Here we show that the tumour suppressor protein LIMD1 (LIM domain-containing protein) acts as a molecular scaffold, simultaneously binding the PHDs and VHL, thereby assembling a PHD-LIMD1-VHL protein complex and creating an enzymatic niche that enables efficient degradation of HIF-1α. Depletion of endogenous LIMD1 increases HIF-1α levels and transcriptional activity in both normoxia and hypoxia. Conversely, LIMD1 expression downregulates HIF-1 transcriptional activity in a manner depending on PHD and 26S proteasome activities. LIMD1 family member proteins Ajuba and WTIP also bind to VHL and PHDs 1 and 3, indicating that these LIM domain-containing proteins represent a previously unrecognized group of hypoxic regulators.

Loss of heterozygosity (LOH) and homozygous deletions at chroLIM proteins ͉ retinoblastoma ͉ K-Ras G12D ͉ epigenetic silencing ͉ tumor suppressor genes