A Nucleolar PUF RNA-binding Protein with Specificity for a Unique RNA Sequence [Plant Biology]

October 20th, 2015 by Zhang, C., Muench, D. G.

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, and these 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 non-classical 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 ten 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, as all natural PUFs studied to date bind to RNAs that contain a conserved UGU sequence at their 5[prime] end and lack specificity for cytosine. Gel mobility shift assays validated the identity of the APUM23 binding sequence and supported the location of three of the ten predicted Puf repeats in APUM23, including the cytosine-binding repeat. The preferred ten-nucleotide sequence bound by APUM23 is present within the 18S rRNA sequence, supporting the known role of APUM23 in 18S rRNA processing. 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.