BIOLOGICAL OVERVIEW

Dosage compensation (compensation for having different numbers of X chromosomes) is a crucial developmental process: without it, females would have twice the amount of X-linked gene product as males, because females have two X chromosomes to a single X chromosome for males. In Drosophila equalization of the amounts of gene product produced by X-linked genes in the two sexes is achieved by hypertranscription of the single male X chromosome. This process is controlled by a set of male-specific lethal (msl) genes that appear to act at the level of chromatin structure. Four proteins (encoded by the male-specific lethal genes) are required for dosage compensation; they associate with the X chromosome in males but not in females. The sequences of the MSL proteins contain several motifs potentially associated with transcriptional control.

• Msl-1 contains an acidic N-terminal domain.
• Msl-2 is a RING finger family member possessing a cysteine-rich zinc-binding motif found in a number of proteins involved in transcriptional regulation, DNA recombination and DNA repair.
• Msl-3 contains two chromo domains, another motif associated with chromosomal proteins involved in transcriptional control.
• Maleless protein (Mle) is the only dosage compensation regulator with defined biochemical activities.

Mle is highly homologous to human RNA helicase A and the bovine counterpart of RNA helicase A, nuclear helicase II, proteins implicated in the unwinding of RNA (Lee, 1997).

Clues to an understanding of the role of Mle in dosage compensation come from three sources: biochemical analysis, genetic analysis, and information about physical interaction of Mle with polytene chromosomes. Mle protein has been shown to possess RNA/DNA helicase activity, adenosine triphosphatase (ATPase) activity and single-stranded (ss) RNA/ssDNA binding activities. Helicases are DNA and RNA unwinding proteins. The helicase activity demonstrates a degree of substrate specificity. Mle displaces substrates containing single stranded RNA regions more efficiently than substrates containing single stranded DNA regions. A mutant of mle (mle-GET) was created that contains a glutamic acid in place of lysine in the conserved ATP binding site A. In vitro biochemical analysis shows that this mutation abolishes both ATPase and helicase activities of MLE but affects the ability of MLE to bind to single stranded DNA and RNA less severly. In vivo, Mle-GET protein can still localize to the male X chromosome but fails to complement mle1 mutant males. These results indicate that the ATPase/helicase activities are essential functions of Mle for dosage compensation but that the interaction of Mle with chromosomes is largely independent of the helicase activity. In other words, the role of Mle in dosage compensation requires at least two functions: (1) the binding to chromosomes, and (2) a helicase activity (Lee, 1997).

How does Mle protein bind to chromosomes? Association of MLE with the male X chromosome is ribonuclease sensitive. Overexpression of Mle or its carboxyl terminus (which includes glycine-rich repeats) reveals an RNase-sensitive affinity for all chromosome arms. These results suggest that nascent messenger RNA transcripts or a hypothetical RNA component of chromatin plays a critical role in the biochemical mechanism of dosage compensation (Richter, 1996).

Part of the Mle protein structure is a conserved RNA binding motif. One RNA binding domain, termed the double-stranded RNA-binding domain (dsRBD), was first identified as 70 residue repeat motifs in three proteins: dsRNA-dependent (DAI) protein kinase, Xenopus RNA-binding protein xlrbpa and Drosophila maternal effect protein Staufen. Two copies of the domain have been detected in helicases: in mammalian RNA helicase A and in Drosophila Maleless. The helicase domain appears to be insufficient on its own to promote helicase activity and additional RNA-binding capacity must be supplied either as domains adjacent to the helicase domain, as in Mle, or by bound RNA recognizing partners (Gibson, 1994).

What is the hierarchy of gene and protein function in dosage compensation? At the top of the hierarchy is Sex lethal, the master regulator of sex determination in Drosophila. Sex lethal regulates the splicing of Msl-2 producing a functional Msl-2 protein in males only (Kelley, 1995 and 1997). The other three male specific lethals are not differentially spliced, although putative SXL-binding sites are also found in the 3' UTR of a subset of MSL-1 transcripts. Msl-2, spliced into a functional form in males, serves to heighten transcriptional activation of the solitary X chromosome. MSL-2 acts in concert with three partners in this task: Maleless, Male-specific lethal-1 and Male specific lethal-3. It is thought that Msl-2 acts in some manner to promote the binding of the other three factors to chromatin. However, Mle might not interact specifically with chromatin but rather with RNA. Is chromosomal RNA the direct target of Mle in dosage compensation, or does RNA have an unknown role in assembling the male specific lethal gene activation complex to the chromosome? There is no reason to assume these possible roles for Mle are mutually exclusive, and both may be involved.

Chromatin is thought to be the target of the MSL proteins. A specific acetylated isoform of Histone H4, H4Ac16, is also detected predominantly on the male X chromosome. Mle and Msl-1 bind to the X chromosome in an identical pattern and the pattern of H4Ac16 on the X chromosome is largely coincident with that of Mle/Msl-1. H4Ac16 was not detected on the X chromosome in homozygous male specific lethal mutant males, correlating with the lack of dosage compensation in these mutants. These data suggest that synthesis or localization of H4Ac16 is controlled by the dosage compensation regulatory hierarchy. Dosage compensation may involve H4Ac16 function, potentially through interaction with the product of the msl genes (Bone, 1994).

The role of Maleless in the chromosomal activation hierarchy remains unclear. Its DNA helicase activity may contribute to destabilizing chromatin structure, analgous to ATP-dependent nucleosomal disruption by the SWI/SNF and Drosophila NURF complexes (see Imitation SWI and Brahma). Its RNA helicase activity could facilitate transcription by altering the structure of nascent RNA, a process that can stimulate reinitiation and/or elongation. Alternatively, Mle may interact with a hypothetical structural RNA component of the chromosome. There is precedence for such RNA in the existence of X chromosome associated RNA on the X. Interestingly, while Mle is released following RNase treatment, the other male specific lethal proteins remain associated with the chromosome (Richter, 1996). This suggests that the several distinct known biochemical functions of male specific lethal proteins will lend themselves to distinct independent functions in dosage compensation (Lee, 1997).

GENE STRUCTURE

Exons - 5

Bases in 3' UTR - 450

PROTEIN STRUCTURE

Amino Acids - 1293

Structural Domains

Maleless contains seven short segments that define a superfamily of known and putative RNA and DNA helicases. This group includes proteins from E. coli, yeast, Drosophila, mammals, and DNA and RNA viruses. Several have been shown to have DNA or RNA helicase activity, or nucleic acid-dependent ATPase activity. The members of the superfamily share seven distinct conserved segments. Two segments contain the "A" and "B" sites of an NTP binding domain motif; no activity has been assigned individually to the others. Mle contains similarity to each of the conserved segments, including the six amino acids that are invariant throughout the superfamily. The location of similarity within the Mle protein falls in the central portion and spans approximately 360 amino acids, from amino acids 400 to 760 (Kuroda, 1991).

Within the superfamily, several subfamilies of related proteins can be distinguished by two criteria: particular signature motifs [e.g., the sequence DEAD (Asp, Glu, Ala, Asp) in segment II] and a higher overall sequence identity throughout the entire helicase domain. On the basis of these criteria, Mle is a member of a subfamily of putative helicase proteins, the DEAH family. The first described members of this family are three proteins involved in pre-mRNA processing in yeast (PRP2, PRP16 and PRP22). The similarity between Mle and PRP2, PRP16 and PRP22 is extensive throughout the putative helicase domain. Similarity between Mle and DEAH family members extends approximately 100 amino acids beyond the last conserved segment (VI) of the large superfamily. Despite the high overall similarity of Mle to DEAH family members, Mle carries a difference in the proposed signature sequence, with the sequence DEIH rather than DEAH in segment II of the putative ATP-binding motif. Outside of the putative helicase domain, Mle does not show significant sequence similarity to proteins in current data bases. The C-terminal region contains nine imperfect repeats of a glycine-rich sequence. The posterior embryonic determinant Vasa, a member of the superfamily of putative helicases, encodes six copies of a glycine-rich heptad repeat (Kuroda, 1991 and references).

date revised: 15 July 97  

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