Characterization of MLE RNA helicase, a subunit of the Dosage Compensation Complex (DCC) in Drosophila melanogaster

2. Summary In Drosophila melanogaster the transcriptional activity
of the male X chromosome is upregulated to compensate for the
reduced dosage of X-linked genes as compared to the two X
chromosomes in females. This process is mediated by the Dosage
Compensation Complex (DCC), a ribonucleoprotein complex consisting
of five proteins (MSL1, MSL2, MSL3, MOF and MLE) and two non-coding
RNAs (roX1 and roX2). The DCC preferentially localizes on the X
chromosomes in males where it doubles its transcription rate. Two
enzymes are associated with the DCC: the acetyltransferase MOF,
specific for the lysine 16 of H4 (H4-K16), and the DNA/RNA helicase
MLE. Genetic experiments demonstrated that both activities are
required for dosage compensation in male flies. However, the weak
association of MLE to the DCC has complicated its biochemical
analysis and, so far, the involvement of MLE RNA helicase in dosage
compensation has only been demonstrated genetically. Using
different in vivo and in vitro approaches the physical and
functional interactions of MLE with the other MSL proteins and with
the roX RNAs was addressed. Monoclonal antibodies, specifically
recognizing MLE, were raised in rats, offering a new tool for MLE
characterization. By coexpression of the DCC subunits in SF9 cells,
a recombinant complex containing MSL1-2-3, MOF, MLE and the roX2
RNA was reconstituted and purified. A specific integration of roX2
into the DCC could be observed only in the absence of MLE. Non
specific RNA binding properties seemed instead associated to MLE
RNA helicase. Moreover, the purified MSL complex did not affect the
ATPase activity of MLE in the presence or absence of roX2 RNA. In
vitro, MLE showed a preferential association with MSL1 and MSL2 and
MLE interaction with both MSL proteins were not RNA mediated. In
view of these results we suggest that binding to roX2 is not the
main determinant for MLE integration into the DCC complex and
protein-protein interactions might instead contribute to its proper
recruitment to the X chromosome. MLE is a member of the DEAD-box
RNA helicase family and it shares with the other members the same
domain organization. In addition to a central ATPase/helicase
domain, two predicted N-terminal double strand (ds) RNA-binding
motifs (dsRBM1 and dsRBM2) and a predicted C-terminal single strand
(ss) RNA/DNA-binding domain (RGG-box) are also present in MLE
protein. These domains have been extensively characterized in RHA,
human ortholog of MLE, and their RNA binding properties confirmed.
However, it is not known how MLE binds RNA and how the different
RNA binding modules contribute to stimulate its enzymatic
activities. A preferential binding of MLE to dsRNA compared to
ssRNA was shown by binding assays. In addition, changes in the
affinity of MLE for both ssRNA and dsRNA were observed in the
presence of different nucleotides. Deletion mutants of MLE were
produced and purified from insect cells in order to address the
contribution of the different RNA binding domains to MLE enzymatic
activities. By transient expression in Drosophila cells of the same
deletion mutants fused to GFP, the effects of individual domains on
MLE recruitment to the X chromosome were also determined. The
results show that unlike RHA, the dsRB1 and the RGG domains are
dispensable for MLE RNA binding and unwinding, whereas dsRB2 seems
to play the major role in coordinating both activities. However,
the enzymatic activities alone are not sufficient to properly
target MLE to the X chromosome. These results provide new data on
the functional properties of MLE RNA helicase that may help to
elucidate its molecular mechanisms of action.