Identification of cellular mRNA targets for RNA-binding protein Sam68

Abstract

Sam68 (Src-associated in mitosis, 68 kDa), a nuclear RNA-binding protein, has been postulated to play a role in cell-growth control as a modulator of signal transduction and activation of RNA metabolism. Although Sam68 was demonstrated to bind to the UAAA sequences in synthetic oligoribonucleotides and poly(U) homopolymers in vitro, the legitimate cellular mRNA target remained unclear. By using the differential display and cDNA-representational difference analysis techniques, followed by reverse transcription polymerase chain reaction of RNAs co-immunoprecipitated with Sam68 from a HeLa cell lysate, we identified 10 mRNA species that bind in vivo to Sam68 in an RNA-binding domain-dependent manner. Among them, the mRNA species for hnRNP A2/B1 and beta-actin were found to bind prominently in vivo as well as in vitro, suggesting the possible involvement of Sam68 in the post- transcriptional regulation of these genes. Mapping of the Sam68-binding sequence revealed that Sam68 associates with these mRNAs through different nucleotide motifs, UAAA for hnRNP A2/B1 mRNA and UUUUUU for beta-actin mRNA, and that both binding sequences must reside in a loop structure for recognition by Sam68. The results indicated that Sam68 recognizes both the UAAA motif and poly(U) sequences in vivo for binding to cellular target mRNAs.

Neurodegenerative diseases: Quantitative predictions of protein–RNA interactions

“…In agreement with experimental evidence (Itoh et al 2002;Sellier et al 2010), we predict that SAM68 does not interact with CGG repeats (Fig. 3A) or negative controls β-actin 1291-1417 nt ( Fig.…”

“…3C) and hnRNPA2/B1 region 435-1173 nt ( Fig. 3E; Supplemental Table 1), as reported in previous studies (Itoh et al 2002;Sellier et al 2010). The fact that SAM68 does not interact with CGG repeats suggests that other proteins could be involved in its sequestration.…”

Apoptotic-induced cleavage shifts HuR from being a promoter of survival to an activator of caspase-mediated apoptosis

“…The mRNA of heterogenous nuclear ribonucleoprotein A1 (hnRNP A1), a negative control, did not associate with HuR. 31 By scanning the 3 0 -UTR of caspase-9 mRNA, we identified two AREs (ARE1: 1841-1870; ARE2: 1944ARE2: -1988 (Supplementary Figure 2). Gel-shift experiments using radioactive-labeled probes showed that both AREs associate with GST-HuR but not GST alone ( Figure 1E).…”

Structural Basis for Homodimerization of the Src-associated during Mitosis, 68-kDa Protein (Sam68) Qua1 Domain

“…It is involved in a variety of pathways, including insulin and T-cell receptor signaling (4), and plays a key role in cell cycle regulation (5). Sam68 exhibits binding specificity for homopolymeric poly(U) RNA and has been shown to recognize UAAA or UUUA sequences with high affinity as determined by Systematic Evolution of Ligands by EXponential Enrichment (SELEX) and in vivo crosslinking (6,7). Post-translational modifications can regulate Sam68 function by critically affecting the accessibility to RNA (8,9).…”