Mapping snoRNA-target RNA interactions in an RNA binding protein-dependent manner with chimeric eCLIP

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• Small nucleolar RNAs (snoRNAs) are crucial non-coding RNAs involved in ribosome and spliceosome biogenesis.
• Many snoRNAs have uncharacterized targets and functions, and the roles of associated proteins are not fully understood.

Purpose of the Study:

• To comprehensively profile snoRNA-target RNA interactions using an enhanced chimeric crosslinking immunoprecipitation (eCLIP) method.
• To investigate the roles of both core and accessory snoRNA binding proteins in mediating these interactions.

Main Methods:

• Adaptation and application of an enhanced chimeric eCLIP approach.
• Utilizing core and accessory snoRNA binding proteins as baits to capture snoRNA-RNA interactions.
• Analysis of interactions in mouse and human cell lines.

Main Results:

• Confirmed known snoRNA-rRNA and snoRNA-snRNA interactions and identified novel interactions for orphan snoRNAs.
• Demonstrated that accessory proteins like WDR43 and NOLC1 modulate specific snoRNA-target RNA interactions.
• Discovered SNORD89's role in U2 snRNA modification, impacting splicing regulation.

Conclusions:

• The enhanced chimeric eCLIP provides a robust framework for studying snoRNA-target interactions in an RNA binding protein-dependent manner.
• Revealed novel regulatory roles for snoRNAs and their associated proteins in RNA biogenesis and splicing.
• Highlighted the importance of snoRNA-protein complexes in fine-tuning cellular RNA processing.