A Nucleolar PUF RNA-binding Protein with Specificity for a Unique RNA Sequence

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.

Ribosome Biogenesis in Plants: From Functional 45S Ribosomal DNA Organization to Ribosome Assembly Factors

“…nuc1 plants show altered growth and leaf development (Pontvianne et al, 2007), while nuc2 plants display delayed flowering time (Durut et al, 2014). Plants with disrupted Arabidopsis PUM-ILIO23 (APUM23) or G-PATCH DOMAIN PROTEIN1 (GDP1) display growth and developmental defects reminiscent of nuc1 mutants (Abbasi et al, 2010;Zhang and Muench, 2015;Kojima et al, 2018). In addition, apum23/salt hypersensitive9 (sahy9) plants exhibit hypersensitivity to salt mediated by abscisic acid (Huang et al, 2018).…”

Reduced Expression of APUM24, Encoding a Novel rRNA Processing Factor, Induces Sugar-Dependent Nucleolar Stress and Altered Sugar Responses in Arabidopsis thaliana

“…APUM9 controls seed dormancy, functioning as a downstream factor of the dormancy regulatory genes REDUCED DORMANCY5 and DELAY OF GERMINATION6 (Xiang et al, 2014(Xiang et al, , 2016. On the other hand, it was recently suggested that APUM23 functions in rRNA processing by directly binding to a -10-nucleotide sequence in 18S rRNA (Zhang and Muench, 2015).…”

Partner‐specific prediction of RNA‐binding residues in proteins: A critical assessment

“…Computational approaches, including statistical machine learning methods, for predicting RNA‐binding sites in RBPs are increasingly valuable because biophysical characterization of protein‐RNA complexes is difficult . Most existing methods for predicting RNA‐binding residues in RBPs use only the sequence and/or structural features of the RBP, without considering the sequence or structure of its putative RNA partner(s) even though RBPs such as Cas9 proteins, Pumilio and zinc finger proteins are known to contain modular RNA‐binding motifs or domains that recognize specific RNA sequence and/or structural features of their binding partners; Base‐specific hydrogen bonding, electrostatic interactions, and geometric factors influence binding specificity . Reliable computational tools for predicting RNA binding residues in RBPs can both complement and help narrow the focus of biophysical and molecular genetic methods for identifying features critical for recognition in protein‐RNA interactions.…”

“…While roughly half of all RBPs bind preferentially to a particular RNA sequence or structural motif, the rest appear to bind RNA in a “nonspecific” manner . Sequence‐specific RBPs typically recognize their RNA binding partners by forming complex binding surfaces that combine multiple modular RNA‐binding motifs or domains (RBDs), examples include Cas9, Pumilio, and zinc finger proteins . Nonspecific RBPs include translation elongation and initiation factors, and proteins involved in RNA degradation .…”