Mutations in the RNA binding domain of stem-loop binding protein define separable requirements for RNA binding and...

Area of Science:

• Molecular Biology
• Gene Expression Regulation
• RNA Processing

Background:

• Posttranscriptional control of replication-dependent histone genes is vital for cell proliferation.
• Stem-loop binding protein (SLBP) is a key regulator, interacting with histone pre-mRNAs.
• SLBP's RNA binding domain (RBD) mediates interaction with the 3' stem-loop structure.

Purpose of the Study:

• To identify critical amino acids within SLBP's RBD essential for stem-loop RNA binding.
• To elucidate the role of specific RBD residues in U7 small nuclear ribonucleoprotein (snRNP) recruitment.
• To understand SLBP's mechanism in facilitating histone pre-mRNA 3' end processing.

Main Methods:

• Site-directed mutagenesis of conserved amino acids in the SLBP RBD.
• In vitro RNA binding assays to assess SLBP-stem-loop interaction.
• Analysis of U7 snRNP recruitment to histone pre-mRNA.

Main Results:

• Mutations in highly conserved RBD amino acids abolish or significantly reduce SLBP binding to the stem-loop RNA.
• Specific RBD residues are required for U7 snRNP recruitment, independent of direct RNA binding.
• A 20-amino acid region C-terminal to the RBD also influences U7 snRNP recruitment.
• The YDRY motif in the RBD is critical: tyrosines for RNA binding, DR dipeptide for processing.

Conclusions:

• The SLBP RBD directly interacts with both the stem-loop RNA and U7 snRNP.
• This dual interaction is essential for efficient histone pre-mRNA 3' end processing.
• SLBP acts as a crucial scaffold, coordinating RNA binding and protein-protein interactions for gene regulation.