The expression, localisation and interactome of pigeon CRY2

Area of Science:

• Molecular biology
• Neuroscience
• Chronobiology

Background:

• Cryptochromes (CRY) are crucial for circadian rhythms and potential magnetoreceptors.
• Avian species possess multiple cryptochromes, but their functions remain largely unexplored.
• This study focuses on pigeon cryptochrome 2 (clCRY2) in the retina.

Purpose of the Study:

• To investigate the expression, localization, and interaction network of clCRY2 in the pigeon retina.
• To identify novel functions and molecular interactions of clCRY2.
• To elucidate the role of clCRY2 in avian visual systems and circadian regulation.

Main Methods:

• RNA sequencing to identify clCRY2 transcript variants (clCRY2a and clCRY2b).
• Monoclonal antibody generation for immunohistochemical analysis of clCRY2 localization.
• Proteomic analysis to identify clCRY2 interacting partners in the retina.

Main Results:

• Two clCRY2 transcript variants, clCRY2a and a novel splice isoform clCRY2b, were identified.
• clCRY2 mRNA expression was observed in all retinal layers, with clCRY2b enriched in nuclear layers.
• Immunohistochemistry revealed clCRY2a/b localization across retinal layers, particularly at the outer limiting and outer plexiform layers.
• Proteomic analysis identified interactions between clCRY2a/b and circadian proteins (PER2, CLOCK, ARTNL), cell junction proteins, and dynein motor components.

Conclusions:

• clCRY2 is a component of the avian circadian clock within the retina.
• clCRY2 unexpectedly associates with the microtubule cytoskeleton, suggesting a role beyond circadian regulation.
• These findings provide new insights into the multifaceted functions of cryptochromes in avian biology.