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

Area of Science:

• Molecular Biology
• Genomics
• Biochemistry

Background:

• PUF proteins are conserved RNA-binding proteins with modular RNA-binding domains, typically featuring eight Puf repeats.
• Plant PUF protein families exhibit significant variability in Puf repeat number, organization, and sequence.
• Variability in RNA-binding domains may lead to nonclassical RNA target sequence preferences in plant PUFs.

Purpose of the Study:

• To investigate if structural variations in plant PUF RNA-binding domains influence RNA target sequence specificity.
• To identify the novel RNA binding sequence for a nucleolar Arabidopsis PUF protein with an atypical RNA-binding domain.

Main Methods:

• Identification of a novel RNA binding sequence for Arabidopsis PUM23 (APUM23).
• Characterization of the APUM23 binding sequence length, core elements, and nucleotide preferences.
• Validation using gel mobility shift assays to confirm binding and Puf repeat location.

Main Results:

• A 10-nucleotide binding sequence for APUM23 was identified, featuring a UUGA core and a cytosine preference at position 8.
• APUM23's binding characteristics differ from other PUF proteins, which typically bind UGU sequences and lack cytosine specificity.
• Gel mobility shift assays confirmed the APUM23 binding sequence and the involvement of 3 Puf repeats, including the cytosine-binding repeat.

Conclusions:

• The identified 10-nucleotide sequence is present in 18S rRNA, supporting APUM23's role in 18S rRNA maturation.
• APUM23's unique RNA-binding properties suggest potential for Puf repeat engineering and targeted RNA regulation.
• APUM23, as an ortholog of yeast Nop9, offers insights into advanced structural backbones for Puf repeat engineering.