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Updated: Apr 19, 2026
Novel RNA-Binding Proteins Isolation by the RaPID Methodology
Published on: September 30, 2016
A divergent Pumilio repeat protein family for pre-rRNA processing and mRNA localization
Chen Qiu1, Kathleen L McCann2, Robert N Wine1
1Epigenetics and Stem Cell Biology Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709; and.
Abstract:
Pumilio/feminization of XX and XO animals (fem)-3 mRNA-binding factor (PUF) proteins bind sequence specifically to mRNA targets using a single-stranded RNA-binding domain comprising eight Pumilio (PUM) repeats. PUM repeats have now been identified in proteins that function in pre-rRNA processing, including human Puf-A and yeast Puf6. This is a role not previously ascribed to PUF proteins. Here we present crystal structures of human Puf-A that reveal a class of nucleic acid-binding proteins with 11 PUM repeats arranged in an "L"-like shape. In contrast to classical PUF proteins, Puf-A forms sequence-independent interactions with DNA or RNA, mediated by conserved basic residues. We demonstrate that equivalent basic residues in yeast Puf6 are important for RNA binding, pre-rRNA processing, and mRNA localization. Thus, PUM repeats can be assembled into alternative folds that bind to structured nucleic acids in addition to forming canonical eight-repeat crescent-shaped RNA-binding domains found in classical PUF proteins.
Insights
Pumilio (PUF) proteins, known for mRNA binding, can also bind DNA and RNA through alternative structures. This discovery expands the known functions of PUF proteins in nucleic acid processing and localization.
Area of Science:
- Molecular Biology
- Structural Biology
- Genetics
Background:
- Pumilio (PUF) proteins are characterized by an eight-repeat Pumilio (PUM) domain, enabling sequence-specific mRNA binding.
- PUM repeats have been identified in proteins involved in pre-ribosomal RNA (pre-rRNA) processing, such as human Puf-A and yeast Puf6, suggesting novel functions beyond canonical mRNA regulation.
Purpose of the Study:
- To investigate the structural basis and functional implications of PUM repeat-containing proteins in nucleic acid binding and processing.
- To explore the alternative folds and binding mechanisms of PUM repeat proteins beyond the canonical PUF structure.
Main Methods:
- Crystal structure determination of human Puf-A to elucidate its three-dimensional arrangement.
- Biochemical assays to assess the DNA and RNA binding properties of Puf-A.
- Mutagenesis studies in yeast Puf6 to evaluate the role of conserved basic residues in nucleic acid binding and function.
Main Results:
- Human Puf-A exhibits an "L"-shaped structure formed by 11 PUM repeats, distinct from the canonical PUF crescent shape.
- Puf-A engages in sequence-independent interactions with DNA and RNA via conserved basic residues.
- Equivalent basic residues in yeast Puf6 are crucial for its RNA binding, pre-rRNA processing, and mRNA localization activities.
Conclusions:
- PUM repeats can assemble into alternative protein folds, enabling binding to structured nucleic acids.
- This expands the functional repertoire of PUF proteins to include roles in pre-rRNA processing and mRNA localization, mediated by sequence-independent nucleic acid interactions.
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