The human pseudouridine synthase PUS7 recognizes RNA with an extended multi-domain binding surface

Area of Science:

• Biochemistry
• Molecular Biology
• Structural Biology

Background:

• Human pseudouridine synthase PUS7 is crucial for development and brain function.
• PUS7 targets numerous RNAs, but the structural basis for its RNA binding is unknown.
• While target RNAs share a consensus sequence, additional recognition elements are likely involved.

Purpose of the Study:

• To elucidate the structure-function relationship of human PUS7.
• To determine the structural basis for PUS7's RNA binding and substrate specificity.
• To understand the mechanism of PUS7-mediated RNA pseudouridylation.

Main Methods:

• X-ray crystallography to determine the structure of human PUS7 at 2.26 Å resolution.
• Structural modeling to analyze interactions with tRNA.
• Biochemical assays to assess RNA binding affinity and pseudouridylation activity.

Main Results:

• The crystal structure revealed two additional subdomains in human PUS7 compared to its bacterial homolog.
• Structural modeling suggests these subdomains enhance tRNA recognition through increased interactions.
• PUS7 requires the complete tRNA structure, not just the consensus sequence, for efficient pseudouridylation.
• PUS7 exhibits medium affinity for non-modifiable RNAs, potentially aiding substrate screening.
• Modified tRNA shows reduced affinity for PUS7, facilitating product release.

Conclusions:

• Human PUS7 employs a combination of structure-specific and sequence-specific recognition for target RNAs.
• Additional subdomains in human PUS7 are critical for tRNA recognition and binding.
• The dissociation mechanism of modified tRNA from PUS7 is elucidated.
• These findings provide mechanistic insights into PUS7's function in RNA modification.