Alu RNA pseudoknot alterations influence SRP9/SRP14 association

Area of Science:

• Genomics
• Molecular Biology
• RNA Biology

Background:

• The human genome contains over 1 million Alu elements, transcribed into non-coding Alu RNAs.
• These Alu RNAs interact with and are regulated by the SRP9/SRP14 protein heterodimer.
• This interaction depends on a 5' pseudoknot domain within the Alu RNA, stabilized by a U-turn motif.

Purpose of the Study:

• To investigate the structural and functional impact of mutations within the Alu RNA U-turn motif.
• To explore the interaction of a novel short Alu RNA, EB120, with SRP9/SRP14.
• To understand the structural diversity among different Alu RNAs.

Main Methods:

• Site-directed mutagenesis of the BC200 U-turn motif.
• Expression analysis of wild-type and mutant BC200, and EB120 in 18 human cell lines and tissues.
• Biochemical assays to assess SRP9/SRP14 association.
• Small-angle X-ray scattering (SAXS) and atomistic computational structure prediction.

Main Results:

• Mutation of a critical guanosine in the BC200 U-turn motif significantly reduced its expression.
• The short Alu RNA EB120, lacking the canonical U-turn triad, did not associate with SRP9/SRP14.
• SAXS and computational modeling indicated that BC200 and its mutant retain a canonical Alu RNA fold, whereas EB120 lacks this structure.

Conclusions:

• The U-turn motif and its nucleotide triad are crucial for SRP9/SRP14 interaction and potentially for the canonical fold of certain Alu RNAs.
• EB120 represents an Alu RNA variant with a distinct structure and lacking SRP9/SRP14 association.
• This study underscores the significant structural diversity within Alu RNAs and the functional consequences of mutations affecting their structure.