Substitutions in RNA-binding protein Hrp1 map a potential interaction surface with the yeast RNA polymerase II...

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• Anti-termination factors for eukaryotic RNA polymerase II (RNAP II) are crucial for gene expression but remain poorly characterized.
• The yeast nuclear RNA-binding protein Hrp1 (also known as Nab4 and CF1B) was investigated for its potential role as an RNAP II anti-termination factor.

Purpose of the Study:

• To provide genetic evidence for Hrp1 functioning as an RNAP II anti-termination factor.
• To investigate the interaction between Hrp1 and RNAP II during transcription termination.

Main Methods:

• Utilized yeast genetics, including specific mutations in RNAP II (Rpb3-K9E) and genome-wide suppressor screens.
• Analyzed readthrough of Nrd1-Nab3-Sen1-dependent (NNS) terminators and cold-sensitive growth phenotypes.
• Investigated mutations in HRP1, focusing on RNA recognition motifs (RRMs) and C-terminal regions.

Main Results:

• A specific mutation in RNAP II (Rpb3-K9E) caused readthrough of NNS terminators and cold-sensitive growth, suggesting altered interaction with an anti-termination factor.
• Genome-wide and targeted suppressor screens identified mutations in HRP1 that rescued the Rpb3-K9E phenotypes.
• Mutations in Hrp1's RRMs and C-terminal region were found to suppress the RNAP II mutation, indicating their role in Hrp1-RNAP II interaction.

Conclusions:

• Hrp1 acts as a nuclear RNA-binding protein functioning as an RNAP II anti-termination factor in yeast.
• Hrp1 likely binds to the RNAP II elongation complex, promoting transcription elongation and preventing premature termination.
• Mutations in Hrp1 that suppress RNAP II mutations likely weaken Hrp1 binding to RNAP II, thereby compensating for altered RNAP II function at terminators.