Structural and biochemical analyses of the DEAD-box ATPase Sub2 in association with THO or Yra1

Area of Science:

• Molecular Biology
• Structural Biology
• Biochemistry

Background:

• Messenger RNA (mRNA) undergoes cotranscriptional processing and packaging into messenger ribonucleoprotein particles (mRNPs) within the nucleus.
• mRNPs require remodeling before export through the nuclear pore complex.
• In yeast, the DEAD-box ATPase Sub2, assisted by the THO complex, facilitates the loading of the mRNA-binding protein Yra1 onto mRNPs.

Purpose of the Study:

• To elucidate the molecular mechanisms underlying mRNP remodeling during nuclear export.
• To gain structural insights into the interactions between Sub2, THO, and Yra1.

Main Methods:

• Determination of crystal structures of two key complexes: THO-Sub2 and Sub2-ATP analogue-RNA-Yra1-C.
• Analysis of protein-protein and protein-nucleic acid interactions.
• Assay of ATPase activity.

Main Results:

• Crystal structure of the THO hetero-pentamer bound to Sub2 at 6.0 Å resolution revealed a half-open Sub2 conformation.
• Crystal structure of Sub2 with an ATP analogue, RNA, and Yra1-C at 2.6 Å resolution showed Sub2 in a closed conformation.
• Both THO and Yra1-C were found to stimulate Sub2's intrinsic ATPase activity.

Conclusions:

• The THO complex acts as a scaffold, localizing Sub2 to specific sites on nuclear mRNPs.
• Yra1 binding induces a conformational change in Sub2, facilitating its ATPase activity for mRNP remodeling.
• These findings provide a molecular basis for Sub2's role in targeted Yra1 loading and mRNP export.