Proteomic analysis of reaper 5' untranslated region-interacting factors isolated by tobramycin affinity-selection reveals a role for La antigen inreaper mRNA translation

Abstract

Translational control is a key step in gene expression regulation during apoptosis. To understand the mechanisms of mRNA translation of a pro-apoptotic gene, reaper (rpr), we adapted the tobramycin-aptamer technique described by Hartmuth et al. (Proc. Natl. Acad. Sci. USA 2002, 99, 16719-16724) for the analysis of proteins interacting with rpr 5' untranslated region (UTR). We assembled ribonucleoprotein complexes in vitro using translation extracts derived from Drosophila embryos and purified the RNA-protein complexes for mas spectrometry analysis. We identified the proteins bound to the 5' UTR of rpr. One of them, the La antigen, was validated by RNA-crosslinking experiments using recombinant protein and by the translation efficiency of reporter mRNAs in Drosophila cells after RNAinterference experiments. Our data provide evidence of the involvement of La antigen in the translation of rpr and set a protocol for purification of tagged-RNA-protein complexes from cytoplasmic extracts.

Acheron, a novel member of the Lupus Antigen family, is induced during the programmed cell death of skeletal muscles in the moth Manduca sexta

“…La protein binds to MDM2 mRNA and enhances translation, resulting in a subsequent decrease in the levels of the tumor suppressor protein p53 (Trotta et al, 2003). As well, La can modulate the stability and/or translation of other apoptosis-associated proteins such as Reaper (Vazquez-Pianzola et al, 2005) and the inhibitor of apoptosis protein (IAP) (Holcik and Korneluk, 2000). While most of the studies of La function have focused on La itself, it is interesting to speculate that Achn may actually mediate some of the in vivo properties ascribed to La.…”

Alternative Mechanisms to Initiate Translation in Eukaryotic mRNAs

“…Along this idea, riboproteomic approaches have facilitated the identification of various proteins interacting with different IRES elements [134]. Most ITAFs are RNA-binding proteins previously identified as transcription regulators, splicing factors, RNA transport, RNA stability, or translation control proteins [133, 135, 136]. Typical examples of multifunctional proteins that act as ITAFs are the polypyrimidine tract-binding protein (PTB), the poly-r(C) binding protein (PCBP2), the SR splicing factor (SRp20), the far upstream element binding protein 2 (FBP2), the lupus La antigen (La), or Gemin5, among others [136–140].…”

A comprehensive analysis of the La-motif protein superfamily

“…Also, the La protein most probably takes part in the quality-control mechanism of newly synthesized noncoding RNAs such as pre-tRNAs (Copela et al 2006;Huang et al 2006;Kadaba et al 2006). But genuine La proteins are versatile factors that were reported in yeasts, flies, and mammals to bind to certain mRNAs and to influence their translation efficiency (Holcik and Korneluk 2000;Kim et al 2001;Cardinali et al 2003;Trotta et al 2003;Inada and Guthrie 2004;Vazquez-Pianzola et al 2005), their stability, or subcellular localization (McLaren et al 1997;Adilakshmi and Laine 2002;Brenet et al 2005). In addition, several reports established that the HsLa (human genuine La) protein directly binds to several viral RNAs, often enhancing their translation and replication efficiency (Costa-Mattioli et al 2004;Raha et al 2004;Domitrovich et al 2005;Xue et al 2007;Bitko et al 2008) and, at least for some viruses, shielding them from the host's defense systems (Bitko et al 2008).…”