Recognition and binding of the human selenocysteine insertion sequence by nucleolin

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• Eukaryotic cells incorporate selenocysteine (an unusual amino acid) at UGA codons, typically stop signals.
• This process relies on a selenocysteine insertion sequence (SECIS) in the 3'-untranslated region of selenoprotein gene transcripts.
• DNA-binding protein B was previously identified as a factor binding to the SECIS element.

Purpose of the Study:

• To identify additional protein partners involved in selenoprotein translation.
• To elucidate the mechanism by which the UGA codon is translated as selenocysteine.

Main Methods:

• RNA electromobility shift assays (EMSA) were used to study protein-SECIS interactions.
• Mutational analysis of the SECIS element was performed.
• Antibody-based assays and co-sedimentation studies were employed to investigate protein interactions.

Main Results:

• Nucleolin was identified as a protein that binds to the SECIS element from the human glutathione peroxidase gene.
• Nucleolin binding competed with endogenous binding activity and was sensitive to mutations in conserved SECIS elements.
• Antibodies against nucleolin and DNA-binding protein B affected complex formation and co-extraction, respectively.
• Nucleolin and DNA-binding protein B were found to co-sediment in ribosomal extracts, indicating they are part of a larger complex.

Conclusions:

• Nucleolin is a component of the selenoprotein translation complex, partnering with DNA-binding protein B.
• This complex links the SECIS element to the ribosome and coding region, facilitating the translation of UGA as selenocysteine.
• The findings reveal a novel mechanism for decoding UGA stop codons during selenoprotein synthesis.