Abstract
PUF proteins are a conserved group of sequence specific RNA-binding proteins that bind to RNA in a modular fashion. The RNA-binding domain of PUF proteins typically consists of eight clustered Puf repeats. Plant genomes code for large families of PUF proteins that show significant variability in their predicted Puf repeat number, organization, and amino acid sequence. Here we sought to determine whether the observed variability in the RNA-binding domains of four plant PUFs results in a preference for nonclassical PUF RNA target sequences. We report the identification of a novel RNA binding sequence for a nucleolar Arabidopsis PUF protein that contains an atypical RNA-binding domain. The Arabidopsis PUM23 (APUM23) binding sequence was 10 nucleotides in length, contained a centrally located UUGA core element, and had a preferred cytosine at nucleotide position 8. These RNA sequence characteristics differ from those of other PUF proteins, because all natural PUFs studied to date bind to RNAs that contain a conserved UGU sequence at their 5' end and lack specificity for cytosine. Gel mobility shift assays validated the identity of the APUM23 binding sequence and supported the location of 3 of the 10 predicted Puf repeats in APUM23, including the cytosine-binding repeat. The preferred 10-nucleotide sequence bound by APUM23 is present within the 18S rRNA sequence, supporting the known role of APUM23 in 18S rRNA maturation. This work also reveals that APUM23, an ortholog of yeast Nop9, could provide an advanced structural backbone for Puf repeat engineering and target-specific regulation of cellular RNAs.
Highlights
We predict that Arabidopsis PUM23 (APUM23) possesses 10 Puf repeats that are not clustered within the C-terminal region but rather are unevenly positioned throughout the central region of the protein (Fig. 1A)
Our prediction for the number and location of the APUM23 Puf repeats and the identity of their TRMs was based on three lines of evidence
All PUF RNA target sequences studied to date contained a UGU core located at the 5Ј end of the sequence [1]
Summary
Protein Expression and Purification—The PUM-HDs of APUM2 (At2g29190, amino acids 614 –963), APUM6 (At4g25880, amino acids 496 – 861), and APUM12 (At5g56510, amino acids 258 –596), and full-length APUM23 (At1g72320, 731 amino acids using the second translation start site) were PCR-amplified from Arabidopsis thaliana leaf cDNA using primers listed in supplemental Table S1. Reaction buffer containing 50 units of RNase Inhibitor (SUPERase1⁄7In; Ambion) This 5:1 molar ratio of protein:RNA was necessary to initiate enrichment of the RNA in the first two rounds of SELEX, because the APUM proteins did not possess full activity. The resin was washed three times in 400 l of reaction buffer followed by elution of the protein-RNA complexes with 200 l of elution buffer (25 mM Tris-HCl, pH 8.0, 200 mM NaCl, 30 mM reduced glutathione, and 3 mM DTT). The PCR amplifications from cDNA templates for each SELEX cycle were performed in 200-l reactions with 1.875 M SELEX forward and reverse primers, 1 mM dNTPs, 1 mM MgCl2, and 4 units of Platinum Pfx polymerase for 10 –12 thermal cycles (94 °C for 35 s, 55 °C for 35 s, and 68 °C for 30 s) and a 3-min final extension at 68 °C. Models that demonstrated the typical PUF concave structure, Puf repeat ␣-helical structure, and TRM positioning were used for repeat and TRM predictions
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