Characterization of the Interaction between Neuronal RNA-binding Protein HuD and AU-rich RNA

Abstract

Hu proteins have been shown to bind to AU-rich elements (AREs) in the 3 -untranslated region of unstable mRNAs. They can thereby inhibit the decay of labile transcripts by antagonizing destabilizing proteins that target these AU-rich sequences. Here we examine the sequence preferences of HuD to elucidate its possible role in counteracting mRNA decay. Using repeats of the prototype destabilizing sequence UU(AUUU) n AUU, we show that all three HuD RNA-binding domains participate in binding to AU-tracts that can be as short as 13 residues, depending on the position of the remaining As. Removal of the A residues, resulting in a poly(U)-tract, increased the affinity of HuD for RNA, suggesting that the presence of As in destabilizing elements might favor the recruitment of other proteins and/or prevent HuD from binding too tightly to AREs. In vitro selection experiments with randomized RNAs confirmed the preference of HuD for poly(U). RNA binding analysis of the related protein HuB showed a similar preference for poly(U). In contrast, tristetraprolin, an mRNA destabilizing protein, strongly prefers AU-rich RNA. Many labile mRNAs contain U-tracts in or near their AREs. Individual AREs may thus differentially affect mRNA halflife by recruiting a unique complement of stabilizing and destabilizing factors.

Selective Gene Expression in Magnocellular Neurons in Rat Supraoptic Nucleus

“…Among these are general transcription factor IIA (Roeder, 1991;Cortes et al, 1992;Ma et al, 1996), transcription factor E2A (Murre et al, 1989;Melloul et al, 2002), Hu antigen D (Keene, 2001;Perrone-Bizzozero and Bolognani, 2002;Park-Lee et al, 2003), and Sox 17 (Kanai et al, 1996;Kanai-Azuma et al, 2002). It is interesting that these widely expressed, transcriptionally related gene products are preferentially expressed in the SON relative to the hypothalamus, with expression ratios ranging from 3.8 to 5.1 (Table 2), again likely correlating with the high rates of transcription that occur in the SON (Burbach et al, 2001).…”

Flavonoids inhibit the AU-rich element binding of HuC

“…The template plasmid for in vitro transcription of an artificial AU-rich elementcontaining probe, pGEM-3Zf(+)-ARE, was constructed by annealing two synthetic oligonucleotides followed by ligation into the EcoRI and BamHI restriction sites of the pGEM-3Zf(+) vector (Promega, Madison, WI, USA) using the oligonucleotides 5'-AATTGTTATTTATTTATTTATTC-3' (forward) and 5'-G ATCGAATAAATAAATAAATAAC-3' (reverse) (15).…”

The RNA-binding Protein HuR Binds to Acetylcholinesterase Transcripts andRegulates Their Expression in Differentiating Skeletal MuscleCells

“…These proteins contain three RNA recognition motifs that are known to bind AREs: the AUUUA general sequence, U-rich sequences, and CU-rich motifs (55,56). The function of this family of mRNA-binding proteins is to stabilize transcripts by inhibiting the de-adenylation and targeting of the transcripts to the exosome or by blocking the activity of specific endonucleases that recognize the AREs (57-59).…”