XAP5 CIRCADIAN TIMEKEEPER regulates RNA splicing and the circadian clock by genetically separable pathways

Area of Science:

• Plant molecular biology
• Circadian rhythms
• RNA splicing

Background:

• The plant circadian clock synchronizes biological activities with daily environmental cycles.
• Post-transcriptional regulators, like splicing factors, influence circadian clock pace.
• The molecular functions of the Arabidopsis circadian regulator XAP5 CIRCADIAN TIMEKEEPER (XCT) are largely unknown.

Purpose of the Study:

• To elucidate the molecular mechanisms by which XCT regulates the Arabidopsis circadian clock.
• To investigate the role of XCT in pre-mRNA splicing and its connection to circadian regulation.

Main Methods:

• Yeast three-hybrid assays to detect protein interactions.
• PacBio Iso-Seq for transcriptome-wide analysis of pre-mRNA splicing.
• Genetic analysis using Arabidopsis mutants (xct, prp19a, prp19b).

Main Results:

• XCT physically interacts with spliceosome components, including the Nineteen Complex (NTC).
• xct mutants exhibit transcriptome-wide pre-mRNA splicing defects, particularly in 3' splice site selection.
• Loss-of-function mutations in NTC components (PRP19A/B) suppress the short period phenotype of xct mutants, but not splicing defects.

Conclusions:

• XCT plays a crucial role in pre-mRNA splicing, with potential circadian regulation of this activity.
• XCT appears to regulate circadian clock function through pathways genetically separable from its splicing role.