Structure-function analysis of the RNA helicase maleless.

Area of Science:

• Genetics
• Molecular Biology
• Developmental Biology

Background:

• Loss of function of the RNA helicase maleless (MLE) in Drosophila melanogaster causes male-specific lethality.
• MLE is essential for X chromosome dosage compensation, a process that equalizes gene expression between male and female flies.
• MLE's role in incorporating non-coding roX RNA into the dosage compensation complex (DCC) and targeting it to the X chromosome is poorly understood.

Purpose of the Study:

• To experimentally verify the RNA-binding characteristics of conserved motifs within MLE.
• To determine the contribution of these motifs to MLE's ATPase and helicase activities.
• To investigate the role of MLE motifs in its nuclear and X chromosome targeting.

Main Methods:

• Sequence comparison to predict RNA-binding domains in MLE.
• Experimental evaluation of RNA-binding characteristics of conserved motifs.
• Assays to measure RNA-stimulated ATPase and helicase activity.
• Analysis of MLE targeting to the nucleus and X chromosome territory.

Main Results:

• The RB2 domain is identified as the primary conditional RNA-binding module essential for ATPase and helicase activity.
• The N-terminal RB1 motif does not bind RNA but is critical for targeting MLE to the X chromosome.
• The C-terminal domain, with a glycine-rich heptad repeat, may contribute to dimerization and RNA binding but is not required for helicase activity.

Conclusions:

• RB2 is the key RNA-binding domain for MLE's enzymatic activities in dosage compensation.
• Distinct domains of MLE have specialized functions: RB2 for activity, RB1 for targeting.
• Understanding MLE's domain functions provides insight into the mechanisms of X chromosome dosage compensation.