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:

• Antitermination 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 as a potential RNAP II antitermination factor.

Purpose of the Study:

• To provide genetic evidence for Hrp1 functioning as an RNAP II antitermination factor.
• To elucidate the mechanism by which Hrp1 interacts with RNAP II and influences transcription termination.

Main Methods:

• Genetic analysis using yeast strains with specific mutations in RNAP II (Rpb3-K9E) and Hrp1.
• Genome-wide suppressor screens to identify mutations that rescue phenotypes associated with Rpb3-K9E.
• Nanopore direct RNA sequencing (direct RNA-seq) to analyze endogenous terminator readthrough.

Main Results:

• A specific mutation in RNAP II (Rpb3-K9E) caused readthrough of Nrd1-Nab3-Sen1-dependent (NNS) terminators and cold-sensitive growth.
• Suppression of Rpb3-K9E phenotypes was achieved by mutations in Hrp1, particularly in its RNA recognition motifs (RRMs).
• Direct RNA-seq confirmed increased readthrough of endogenous NNS terminators in Rpb3-K9E mutants and suppression by Hrp1 mutations.

Conclusions:

• Hrp1 functions as an RNAP II antitermination factor, promoting transcription elongation.
• Hrp1 likely binds to the RNAP II elongation complex and is less readily released at terminators when RNAP II is mutated (Rpb3-K9E).
• Mutations in Hrp1 that suppress Rpb3-K9E likely weaken its interaction with RNAP II, restoring proper termination regulation.