Analysis of CUGBP1 Targets Identifies GU-Repeat Sequences That Mediate Rapid mRNA Decay

Abstract

CUG-repeat binding protein 1 (CUGBP1) mediates selective mRNA decay by binding to GU-rich elements (GREs) containing the sequence UGUUUGUUUGU found in the 3 untranslated region (UTR) of short-lived transcripts. We used an anti-CUGBP1 antibody to immunoprecipitate CUGBP1 from HeLa cytoplasmic extracts and analyzed the associated transcripts using oligonucleotide microarrays. We identified 613 putative mRNA targets of CUGBP1 and found that the UGUUUGUUUGU GRE sequence and a GU-repeat sequence were both highly enriched in the 3 UTRs of these targets. We showed that CUGBP1 bound specifically to the GU-repeat sequence and that insertion of this sequence into the 3 UTR of a beta-globin reporter transcript conferred instability to the transcript. Based on these results, we redefined the GRE to include this GU-repeat sequence. Our results suggest that CUGBP1 coordinately regulates the mRNA decay of a network of transcripts involved in cell growth, cell motility, and apoptosis.

Regulation of CUG-binding Protein 1 (CUGBP1) Binding to Target Transcripts upon T Cell Activation

“…We found that several GRE-like sequences were the most highly enriched motifs (Table 2, left). These results corroborated previous reports showing that both (UGUU) n motifs and (UG) n motifs were enriched in the 3Ј-UTRs of CUGBP1 target transcripts (13,25). Additionally, the 3Ј-UTRs from CUGBP1 target transcripts were submitted to a BioProspector motif search (26) to evaluate enriched sequences.…”

“…RNA-IP and Microarray Analysis-Cytoplasmic extracts were prepared from resting and stimulated T cells, and RNA-IP reactions were performed as described previously, using antibodies targeting hemagglutinin (HA) (F7, Santa Cruz Biotechnology, Inc. (Santa Cruz, CA)), CUGBP1 (3B1, Santa Cruz Biotechnology, Inc.) or poly(A)-binding protein (PABP) (ImmunoQuest) (13). Three RNA-IP experiments were performed using resting or activated T cells isolated from three individual anonymous donors.…”

“…We recently showed that the GRE was highly enriched in short lived transcripts expressed in primary human T cells (12) and that CUGBP1 bound to the GRE and mediated the decay of GRE-containing transcripts (13). The transcriptional network targeted by CUGBP1 and the regulation of CUGBP1 function in T cells remain poorly understood.…”

“…Transcripts encoding RNAbinding proteins were particularly enriched in the CUGBP1 targets, suggesting that in resting T cells, CUGBP1 may be functioning as a posttranscriptional "regulator of regulators" whereby CUGBP1 influences the expression of a network of target transcripts encoding RNA-binding proteins, which in turn regulate individual subnetworks of transcripts. A partial list of important CUGBP1 target transcripts is shown in Table 1, and a full list can be found in supplemental Table 1. CUGBP1 binds preferentially to a GU-rich RNA motif, UGU(G/U)UGU(G/U)UGU, found in the 3Ј-UTR of target transcripts (13). We sought to determine whether our experimentally determined CUGBP1 target transcripts in T cells were enriched for this motif.…”

“…The GRE was originally identified as a sequence that was highly enriched in the 3Ј-UTR of transcripts that decayed rapidly in primary human T cells and was shown to function as a regulator of mRNA decay (12). Based on a bioinformatic analysis of mRNA targets of CUGBP1 in HeLa cells, the GRE was defined to be the consensus sequence UGU(G/U)UGU(G/U)UGU (13). Binding by CUGBP1 to certain GRE-containing transcripts has been shown to promote their rapid degradation, but how this process is regulated during cellular activation is poorly understood.…”